The mitogen-activated protein kinase（MAPK） signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosp...The mitogen-activated protein kinase（MAPK） signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosphatase 1（MKP1） has an inhibitory effect on the p38 MAPK and JNK pathways, but it is unknown whether it plays a role in Aβ-induced oxidative stress and neuronal inflammation. In this study, PC12 cells were infected with MKP1 sh RNA, MKP1 lentivirus or control lentivirus for 12 hours, and then treated with 0.1, 1, 10 or 100 μM amyloid beta 42（Aβ42）. The cell survival rate was measured using the cell counting kit-8 assay. MKP1, tumor necrosis factor-alpha（TNF-α） and interleukin-1β（IL-1β） m RNA expression levels were analyzed using quantitative real time-polymerase chain reaction. MKP1 and phospho-c-Jun N-terminal kinase（JNK） expression levels were assessed using western blot assay. Reactive oxygen species（ROS） levels were detected using 2′,7′-dichlorofluorescein diacetate. Mitochondrial membrane potential was measured using flow cytometry. Superoxide dismutase activity and malondialdehyde levels were evaluated using the colorimetric method. Lactate dehydrogenase activity was measured using a microplate reader. Caspase-3 expression levels were assessed by enzyme-linked immunosorbent assay. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase d UTP nick end labeling method. MKP1 overexpression inhibited Aβ-induced JNK phosphorylation and the increase in ROS levels. It also suppressed the Aβ-induced increase in TNF-α and IL-1β levels as well as apoptosis in PC12 cells. In contrast, MKP1 knockdown by RNA interference aggravated Aβ-induced oxidative stress, inflammation and cell damage in PC12 cells. Furthermore, the JNK-specific inhibitor SP600125 abolished this effect of MKP1 knockdown on Aβ-induced neurotoxicity. Collectively, these results show that MKP1 mitigates Aβ-induced apoptosis, oxidative stress and neuroinflammation by inhibiting the JNK signaling pathway, thereby playing a neuroprotective role.展开更多
Objective:The aim of the study was to investigate the effect of c-Jun N-terminal protein kinase(JNK) signaling pathway on influencing the sensitivity to radiotherapy of human nasopharyngeal carcinoma CNE cells.Methods...Objective:The aim of the study was to investigate the effect of c-Jun N-terminal protein kinase(JNK) signaling pathway on influencing the sensitivity to radiotherapy of human nasopharyngeal carcinoma CNE cells.Methods:Human nasopharyngeal carcinoma CNE multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Western blot was employed to analyze the activity of JNK signaling pathway in MCS after X-ray irradiation,and the expression of caspase-3 protein before and after using SP600125(a special inhibitor of JNK).X-ray induced cell apoptosis in MCS before and after treated with SP600125 were detected by TUNEL.Results:The level of JNK phosphorylation in MCS was a dynamic course after radiation,and there was a phosphorylation peaks at 2 h later,the apoptotic rate of MCS(P < 0.05) and the expression of caspase-3 protein(P < 0.05) were significantly increased after treated with SP600125.Conclusion:The transient activation of JNK played a important role in sensitivity to radiotherapy of CNE MCS via mediating survival signals,blocking this pathway accelerate cell apoptosis,which may be related to the increased expression of caspase-3.展开更多
[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signal...[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.展开更多
文摘The mitogen-activated protein kinase（MAPK） signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosphatase 1（MKP1） has an inhibitory effect on the p38 MAPK and JNK pathways, but it is unknown whether it plays a role in Aβ-induced oxidative stress and neuronal inflammation. In this study, PC12 cells were infected with MKP1 sh RNA, MKP1 lentivirus or control lentivirus for 12 hours, and then treated with 0.1, 1, 10 or 100 μM amyloid beta 42（Aβ42）. The cell survival rate was measured using the cell counting kit-8 assay. MKP1, tumor necrosis factor-alpha（TNF-α） and interleukin-1β（IL-1β） m RNA expression levels were analyzed using quantitative real time-polymerase chain reaction. MKP1 and phospho-c-Jun N-terminal kinase（JNK） expression levels were assessed using western blot assay. Reactive oxygen species（ROS） levels were detected using 2′,7′-dichlorofluorescein diacetate. Mitochondrial membrane potential was measured using flow cytometry. Superoxide dismutase activity and malondialdehyde levels were evaluated using the colorimetric method. Lactate dehydrogenase activity was measured using a microplate reader. Caspase-3 expression levels were assessed by enzyme-linked immunosorbent assay. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase d UTP nick end labeling method. MKP1 overexpression inhibited Aβ-induced JNK phosphorylation and the increase in ROS levels. It also suppressed the Aβ-induced increase in TNF-α and IL-1β levels as well as apoptosis in PC12 cells. In contrast, MKP1 knockdown by RNA interference aggravated Aβ-induced oxidative stress, inflammation and cell damage in PC12 cells. Furthermore, the JNK-specific inhibitor SP600125 abolished this effect of MKP1 knockdown on Aβ-induced neurotoxicity. Collectively, these results show that MKP1 mitigates Aβ-induced apoptosis, oxidative stress and neuroinflammation by inhibiting the JNK signaling pathway, thereby playing a neuroprotective role.
文摘Objective:The aim of the study was to investigate the effect of c-Jun N-terminal protein kinase(JNK) signaling pathway on influencing the sensitivity to radiotherapy of human nasopharyngeal carcinoma CNE cells.Methods:Human nasopharyngeal carcinoma CNE multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Western blot was employed to analyze the activity of JNK signaling pathway in MCS after X-ray irradiation,and the expression of caspase-3 protein before and after using SP600125(a special inhibitor of JNK).X-ray induced cell apoptosis in MCS before and after treated with SP600125 were detected by TUNEL.Results:The level of JNK phosphorylation in MCS was a dynamic course after radiation,and there was a phosphorylation peaks at 2 h later,the apoptotic rate of MCS(P < 0.05) and the expression of caspase-3 protein(P < 0.05) were significantly increased after treated with SP600125.Conclusion:The transient activation of JNK played a important role in sensitivity to radiotherapy of CNE MCS via mediating survival signals,blocking this pathway accelerate cell apoptosis,which may be related to the increased expression of caspase-3.
基金Supported by National Natural Science Foundation of China(81760806)Project of Traditional Chinese Medicine Administration of Gansu Province(GZK-2019-28)Innovation Ability Improvement Project of Higher Education Institutions of Gansu Province(2019B-103)。
文摘[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.
文摘目的通过比较瘢痕疙瘩及正常皮肤中细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、应激活化蛋白激酶(c-Jun amino-terminal kinase,JNK)信号通路的表达,探讨其在瘢痕疙瘩形成中的作用。方法取四川大学华西医院烧伤整形科收治的26例患者皮肤组织,其中行瘢痕疙瘩切除患者(实验组)16例,瘢痕疙瘩形成时间8个月～10年;胸部6例,耳垂4例,会阴部2例,肩部3例,腹部1例;均经病理检查确诊为瘢痕疙瘩。10例整形手术患者自愿捐赠的正常皮肤作为对照组,腹部4例,大腿3例,肩部2例,背部1例。取标本采用Envision二步法行免疫组织化学染色,观察磷酸化及非磷酸化JNK和ERK表达情况,并采用Image Pro Plus 4.5图像分析系统测定积分吸光度(IA)值,观察阳性染色强度。结果免疫组织化学染色观察显示,对照组正常皮肤成纤维细胞中未见明显的磷酸化及非磷酸化ERK、JNK阳性表达;而实验组主要在成纤维细胞中表达,阳性颗粒主要位于细胞质和细胞核内。实验组磷酸化ERK及JNK的IA值明显高于对照组(P<0.05),非磷酸化ERK及JNK两组间差异无统计学意义(P>0.05)。结论磷酸化JNK、ERK信号通路蛋白在瘢痕疙瘩中异常高表达,提示该通路可能与瘢痕疙瘩形成密切相关。
