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Aim To investigate the molecular mechanism of the protection of vascular smooth muscle cells by the regulation of endoplasmic reticulum stress and autophagy by Fufang Danshen dripping pills.Methods Fifty patients with atherosclerosis were randomly divided into two groups; one group received normal treatment,while the other group was added Fufang Danshen dripping pills,and the clinical efficacy was observed.Vascular smooth muscle cells were divided into control,ox LDL model,Fufang Danshen dripping pill group,Fufang Danshen dripping pill+endoplasmic reticulum stress inhibitor group,and Fufang Danshen dripping pill+endoplasmic reticulum stress inducer group.Proliferation was detected,and vasodilator function factors and oxidative stress were measured in each group,ER stress marker proteins,autophagy marker proteins and apoptosis related protein expression were detected,and activation of the IRE1-TRAF2-ASK1-JNK signaling pathway was detected.Results Compared with control group,various indicators of cells in ox-LDL model group showed that they were under ER stress,high oxidative stress,high autophagy status,and the IRE1-TRAF2-ASK1-JNK pathway was found to be over activated.However,compared with ox LDL model group,the above indicators in Fufang Danshen dripping pill group and Fufang Danshen dripping pill+endoplasmic reticulum stress inhibitor group were significantly better,the IRE1-TRAF2-ASK1-JNK pathway was over activated,and the endoplasmic reticulum stress inhibitor was more obvious,and Fufang Danshen dripping pill+endoplasmic reticulum stress inducer group significantly reversed the improved effects of Fufang Danshen dripping pills.Conclusions Fufang Danshen dripping pills protect vascular smooth muscle cells by inhibiting excessive activation of the IRE1-TRAF2-ASK1-JNK pathway,decreasing endoplasmic reticulum stress,maintaining proper autophagy,and inhibiting abnormal cell proliferation and apoptosis.
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BACKGROUND: Studies have shown that disulfiram has anti-tumor activity, which can be combined with copper (Cu) ions to exert an anti-tumor effect on multiple tumors in vivo and in vitro, but the effect of disulfiram on osteosarcoma proliferation and apoptosis has not been clarified. OBJECTIVE: To investigate the effect of disulfiram combined with Cu on osteosarcoma proliferation and apoptosis and the possible mechanism in vivo and in vitro. METHODS: The study protocol was approved by the Animal Ethics Committee of Shanxi Medical University (approval No. 2017LL077). (1) In vitro study: Diethyldithiocarbamate (DDTC)-Cu (0.5, 1, 2, 3, and 5 μmol/L) was configured with Cu and DDTC which is the transformation of disulfiram after absorbed by bodies. DDTC single drug (5 μmol/L), Cu single drug (5 μmol/L) and blank control groups were set. Osteosarcoma cell lines Saos-2 and MG-63 were treated with drugs, and cell counting kit-8 assay was used to detect the inhibitory effect of DDTC-Cu at different concentrations on the proliferation of Saos-2 and MG-63 cells. Changes in Saos-2 apoptosis were measured by AnnexinV-FITC/PI staining. (2) In vivo study: A total of 10 BALB/c-nu/nu female nude mice of 4 weeks old were randomly divided into DDTC-Cu group and control group. The mixture of Saos-2 cells and Matrigel (1:1 mixed, 400 μL per mouse) was injected subcutaneously into the right back of nude mice. Two weeks after inoculation, model mice were intraperitoneally injected dexamethasone (0.5 mg/kg once every other day) in the control group, and dexamethasone (0.5 mg/kg once every other day) and DDTC-Cu complex (10 nmol/g once every other day) in the DDTC-Cu group. Xenograft tumors in each group were measured at regular intervals and tumor growth curves were drawn. Five weeks after inoculation, the animals were sacrificed under anesthesia, and tumors were completely removed. Immunohistochemistry was used to detect the expression level of ki67 protein in tumor paraffin sections. The expressions of proteins related to cell proliferation and apoptosis and JNK pathway proteins were determined by western blot analysis. RESULTS AND CONCLUSIONS: (1) In vitro study: The proliferation inhibition in the DDTC-Cu group was significantly stronger than that in the DDTC single drug, Cu single drug and blank control groups. Cell counting kit-8 results showed that DDTC-Cu inhibited osteosarcoma proliferation in a dose-dependent manner, with 50% inhibiting concentration of 0.337 μmol/L (Saos-2) and 0. 487 μmol/L (MG-63) for 24 hours, respectively. The results of flow cytometry showed that DDTC-Cu promoted Saos-2 apoptosis in a dose-dependent manner. (2) In vivo study: The tumor volume and mass of the DDTC-Cu group were smaller than those of the control group. Immunohistochemical results showed that the expression level of ki-67 protein in the DDTC-Cu group was lower than that in the control group. Western blot results showed that the expression levels of p-JNK and c-jun in the DDTC-Cu group were up-regulated. To conclude, disulfiram combined with Cu inhibits proliferation and induces apoptosis of osteosarcoma in vitro and in vivo, and its mechanism may be related with activation of JNK signaling pathway.
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Obj ective To investigate the mechanism of secreted frizzled-related Protein 5 ( sFRP5) expression in cardiomyocyte hypertrophy.Methods In vivo experiment, neonatal rat ventricular myocytes were exposed to Ang Ⅱ(10-6 mmol/L, 48 h).Telmisartan, Y27632, PD98059,SB203580 and SP600125 were used to block angiotensin type 1 receptor( AT1R) , Rho/ROCK, p38 MAPK, ERK1/2 and JNK pathway, respective-ly.Western blot was applied to determine the expressions of sFRP5, ROCK1, ROCK2, total MYPT1, p-MYPT1.RT-PCR was used to determine sFRP5 expression.Results There was significant inhibition of sFRP5 expression when treated with Y37632 and SP699125, but less with SB203580 and PD98059 in AngⅡ-induced cardiomyocytes.Moreover, telmisartan down-regulated the expression of ROCK1, but no effect on the expression of ROCK2.Conclusions The expression of sFRP5 is up-regulated mainly by Rho/ROCK1/JNK pathway in cardiomyocyte hypertrophy induced by Ang Ⅱ.
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Andrographolide (ANDRO) has been studied for its immunomodulation, anti-inflammatory, and neuroprotection effects. Because brain hypoxia is the most common factor of secondary brain injury after traumatic brain injury, we studied the role and possible mechanism of ANDRO in this process using hypoxia-injured astrocytes. Mouse cortical astrocytes C8-D1A (astrocyte type I clone from C57/BL6 strains) were subjected to 3 and 21% of O2 for various times (0-12 h) to establish an astrocyte hypoxia injury model in vitro. After hypoxia and ANDRO administration, the changes in cell viability and apoptosis were assessed using CCK-8 and flow cytometry. Expression changes in apoptosis-related proteins, autophagy-related proteins, main factors of JNK pathway, ATG5, and S100B were determined by western blot. Hypoxia remarkably damaged C8-D1A cells evidenced by reduction of cell viability and induction of apoptosis. Hypoxia also induced autophagy and overproduction of S100B. ANDRO reduced cell apoptosis and promoted cell autophagy and S100B expression. After ANDRO administration, autophagy-related proteins, S-100B, JNK pathway proteins, and ATG5 were all upregulated, while autophagy-related proteins and s100b were downregulated when the jnk pathway was inhibited or ATG5 was knocked down. ANDRO conferred a survival advantage to hypoxia-injured astrocytes by reducing cell apoptosis and promoting autophagy and s100b expression. Furthermore, the promotion of autophagy and s100b expression by ANDRO was via activation of jnk pathway and regulation of ATG5.
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Animals , Mice , Astrocytes/drug effects , Autophagy/drug effects , Cell Hypoxia/drug effects , Diterpenes/pharmacology , S100 Calcium Binding Protein beta Subunit/drug effects , Apoptosis/drug effects , Astrocytes/physiology , Blotting, Western , Cell Survival/drug effects , Real-Time Polymerase Chain Reaction , S100 Calcium Binding Protein beta Subunit/metabolism , Time Factors , TransfectionABSTRACT
Objective To analyze the protective effects ofbutylphthalide(NBP) on apoptosis factors (p-JNK,Fas and FasL) of death receptor pathway in JNK pathway of cell model of Parkinson's disease (PD).Methods SH-SY5Y cell apoptosis model induced by MPP + was established in vitro.The cells were divided into four groups:normal control group,SHSYSY cells were treated with complete medium without drug intervention;MPP+ group,1 mmol·L-1 MPP+ was added into the cells;NBP+ MPP+ group,the cells were pretreated with 10 mol·L-1 NBP for 3 h and added with 1 mmol·L-1 MPP+;SP600125 + MPP+ group,the cells were cultured with 10 mol·L-1 JNK inhibitor SP600125 pretreatment for 3 h and 1 mmol·L-1 MPP+ was added.The proliferative potentiality of SH-SY5Y cells induced by MPP+ was measured by MTT.The apoptotic rate was analyzed by Annexin-V/PI (FCM).The morphology of SH-SY5Y cells was observed by inverted phase contrast microscope.The expression of apoptotic protein p-JNK,Fas,FasL was detected by Western blotting.Results The cell proliferative potentiality in the MPP+ group (49.30 ± 2.07)％ was significantly lower than that of the normal control group (100.00 ±0.00)％ (P ＜ 0.05).The cell proliferative potentiality in NBP + MPP + group and SP600125 + MPP + group were (71.90 ±2.10) ％ and (76.40 ± 2.80) ％,which was significantly higher than that of the MPP + group (P ＜ 0.05).Apoptosis rate in the MPP + group (32.27 ± 2.26) ％ was significantly higher than that of the normal control group (10.63 ± 2.07) ％ (P ＜ 0.05).The apoptosis rate in the NBP + MPP + group and SP600125 + MPP+ group were (21.13 ± 3.63) ％ and (19.15 ± 2.63) ％,and the apoptosis rate was significantly lower than that in the MPP+ group(P ＜0.05).The protein expression levels of p-JNK,Fas and FasL were significandy lower in NBP + MPP+ group and SP600125 + MPP+ group than that in the MPP+ group (P ＜0.05).Conclusion Butylphthalide can protect the injury of SH-SY5Y cells induced by MPP+.The mechanism of butylphthalide inhibiting apoptosis may be achieved through regulating p-JNK,Fas and FasL protein expression of death receptor pathway in JNK pathway and inhibiting the cell apoptosis.
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Mitogen-activated protein kinase (MAPK) signaling pathways may be involved in various activities of cells such as apoptosis,differentiation and autophagy.In human cells,MAPK signaling pathways mainly contain Erk,p38 MAPK and JNK,and each of them is highly specific and has its own function.MAPK signaling pathways are closely related to the development of leukemia.The effect of MAPK signaling pathways on leukemia cells is mainly mediated by affecting cell apoptosis,cell drug resistant,cell autophagy and differentiation.In this review,recent advances on the study of MAPK in leukemia are reviewed.
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Aim To observe whether the activation of aldehyde dehydrogenase 2 ( ALDH2 ) can protect a-gainst diabetes induced liver injury in rat model, and analyze the role of JNK pathway in the liver protection induced by activation of ALDH2 . Methods All male SD rats were randomly divided into three groups: nor-mal control group ( Con ) , diabetes group ( DM ) and ethanol + diabetes group ( EtOH + DM ) . After 8 weeks, the fasting blood glucose ( FBG) level, glyco-sylated hemoglobin ( HbA1c) level, serum AST and ALT levels were measured. The changes of hepatic pa-thology were observed by hematoxylin and eosin ( HE) staining method. The protein expressions of ALDH2, JNK and p-JNK in liver tissue were measured. Result Compared with control group, in DM group, the lev-els of FBG and HbA1c, serum AST and ALT levels were increased significantly. The structure of liver mor-phology was destroyed, disarranged and unclear, the hepatocyte was swollen, and a large number of inflam-matory cells were infiltrated. ALDH2 protein expres-sion was decreased, while the expressions of JNK, p-JNK and the ratio of p-JNK/JNK were increased. Com-pared with DM group, in EtOH+DM group, the levels of FBG and HbA1c, serum AST and ALT levels were decreased. The expression of ALDH2 protein was in-creased, accompanying with the decrease of JNK, p-JNK protein expressions and the ratio of p-JNK/JNK. Conclusion Activation of ALDH2 can protect the liv-er against diabetes induced liver damage in rat model, which may be relevant with inhibiting the JNK path-way.
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Objective: To investigate the effect of berbamine (BBM) on the induction of apoptosis in human lung cancer cell A549 cells and the activity of TNF-α-JNK signaling pathway. Methods: After the A549 cells being treated with BBM at different concentration, the inhibitory effect of BBM on proliferation was detected by MTT assay. Cell morphological changes were detected by light microscope. Alteration of apoptosis rate of A549 cells was determined by Annexin V/PI double staining. Western blotting was used to detect the activity of apoptosis-related proteins, including Bcl-x, Caspase-3, and PAPR. The expressions of c-jun N-terminal kinase (JNK) and p-JNK were determined by Western blotting. Changes of tumor necrosis factor-alpha (TNF-α) were detected by fluorescent quantitative PCR and ELISA, respectively. Finally, the impacts of BBM on the proliferation and apoptosis of A549 cells were detected in the absence or presence of a JNK inhibitor. Results: BBM significantly inhibited the growth of A549 cells in a dose-dependent manner. The IC50 of 24 h was 9.01 μmol/L. Cells treated with BBM showed the typically morphological characteristics of apoptotic cells. Annexin V/PI double staining test indicated that BBM could induce the apoptosis of A549 cells in a dose-dependent manner. The early apoptotic population of cells treated with 10 μmol/L BBM was 13.8%, which was 5.6 times higher than that of the control. BBM decreased the expression of anti-apoptotic protein Bcl-x and increased the activity of proapoptotic proteins of caspase-3 and PARP. The mRNA and the protein expression levels of TNF-α were significantly increased by BBM treatment. The p-JNK expression was also dramatically up-regulated after BBM treatment. The effects of BBM on the proliferation and apoptosis in A549 cells were significantly reduced when JNK pathway was blocked. Conclusion: BBM could inhibit the growth and induce the apoptosis of A549 cells, and its mechanism may be related to the activated TNF-α-JNK signaling pathway.
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Background & objectives: Ex vivo expansion of the limbal epithelial cells activates the nerve growth factor (NGF) mediated downstream signal transduction pathway. It is not clear as to which factors control the stemness of the corneal limbal stem cells, i.e., the maintenance of stem cell properties. It is likely that various signaling pathways are involved, including Notch, Wnt and NGF signaling, etc. In the present study, we investigated the activation of phosphoinositide-3-kinase (PI3K) pathway on cells cultured over the chitosan matrix, chitosan silver matrix, chitosan gold matrix, intact and denuded human amniotic membrane (HAM). Methods: Human limbal biopsies obtained from the cadaveric donor eyes were used in this study. The cells cultured over different substrate and observed for the activation of the downstream signaling molecules of PI3K/Akt/FKHRL1 pathway. Western blotting was done to prove the results. Results: The cells cultured over the intact HAM showed the activation of the downstream signaling molecules of PI3K/Akt/JNK pathway compared to the cells grown over other substrates. On inhibition of the PI3K activity there was absence of phosphorylation of downstream effectors in the limbal epithelial cells from the explant culture over the intact HAM. Interpretation & conclusions: The ex vivo expansion of human limbal epithelial progenitor cells on intact HAM was mediated by PI3K/Akt/FKHRL1 pathway, which is known to govern cell survival, and the mitogen-activated protein kinase (MAPK) pathway, known to control the cell mitosis.
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Though the mechanisms involved in the induction of pancreatic islet β cell dysfunction and apoptosis under diabetes conditions are still not clear, previous studies have shown that triggered or aggravated endoplasmic reticulum stress and oxidative stress play essential roles in impairment of β cell functions, especially the JNK pathway which can be activated by both of them.
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AIM: To investigate the expression of tissue factor(TF) induced by oxidized high density lipoprotein(oxHDL) in human umbilical vein cell line,ECV304,and the related mechanisms.METHODS: Four main groups were designed: the negative,the positive(ECV304 with histamine),the HDL group and the oxHDL group.Quantitative real-time polymerase chain reaction(RQ-PCR) and Western blotting were used to detect the expression level of TF.The specific inhibitors of MAPKs,SP600125(c-jun terminal NH2 kinase,JNK),SB203580(p38 MAP kinase,p38 MAPK),PD98059(extracellular signal-regulated kinase,ERK1/2) were used to investigate the underlying mechanisms.RESULTS: The TF expression in normal ECV304 cell line was not detected.Histamine administration resulted in a significant expression of TF in ECV304 cell line,with strongest effect after 1 h co-incubation at concentration of 1?10-5 mol/L histamine(about 4.8-fold higher expression of TF compared with that of 1?10-9 mol/L histamine).Expression level of TF was detected after stimulated with oxHDL in dose-and time-dependent manners.The highest expression of TF mRNA was found at 20 mg/L oxHDL and 6 h co-incubation,with 1.8-fold and 5.3-fold increase in TF expression,respectively,compared with that at 10 mg/L oxHDL and 2 h co-incubation.20 mg/L oxHDL also caused an apparent augmentation of TF protein expression,about 1.5-fold higher compared with that stimulated by 40 mg/L oxHDL.HDL co-incubation did not cause a detectable expression of TF protein.The mRNA levels of TF in ECV304 cell line induced by oxHDL were decreased by 95.0%,81.0%,87.0%,respectively(all P
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Apoptosis is important to the development of diseases. Research recently indicates that c-Jun N-terminal kinase (JNK) and cysteinyl aspartate-specific protease (caspase) play key roles in apoptosis and affect the development of diseases. This article is to introduce the function and relationship between JNK and caspase in apoptosis during the process of diseases.