Regulation of stress-associated scaffold proteins JIP1 and JIP3 on the c-Jun NH2-terminal kinase in ischemia–reperfusion

2010 ◽  
Vol 88 (11) ◽  
pp. 1084-1092 ◽  
Author(s):  
Bing Xu ◽  
Yaling Zhou ◽  
Karmin O ◽  
Patrick C. Choy ◽  
Grant N. Pierce ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Model ◽  
Ischemia Reperfusion ◽  
Scaffold Proteins ◽  
Signaling Cascade ◽  
Interacting Proteins ◽  
H9c2 Cells ◽  
Signaling Specificity ◽  
Sirna Treatment ◽  
A Cell

Ischemia–reperfusion (IR)-induced cell apoptosis involves the activation of c-Jun NH2-terminal kinase (JNK). The activation of JNK requires the presence of scaffold proteins called JNK-interacting proteins (JIP), which bind several members of a signaling cascade for proper signaling specificity. In this study, the expression of scaffold proteins JIP1 and JIP3 and their roles in the regulation of JNK activity were investigated in simulated IR in a cell model (H9c2). JIP1 protein expression was significantly decreased, whereas JIP3 protein expression was increased in IR H9c2 cells. Adenovirus-induced overexpression of JIP1 reduced IR-induced JNK activity and apoptosis. Conversely, overexpression of JIP3 increased JNK activity and apoptosis following IR. Depletion of endogenous JIP1 by siRNA treatment increased the IR-induced JNK activity, whereas siRNA-mediated depletion of endogenous JIP3 inhibited JNK activity. These results suggest that JIP1 and JIP3 play important roles in the activation of JNK during simulated IR challenge in H9c2 cells.

scholarly journals Si-Miao-Yong-An Decoction maintains the cardiac function and protects cardiomyocytes from myocardial ischemia and reperfusion injury

2021 ◽  
Author(s):  
wenwen cui ◽  
Xin Shen ◽  
Lingjuan Zhu ◽  
Mingye Wang ◽  
Yuanyuan Hao ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Protein Expression ◽  
Western Blot ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
H9c2 Cells ◽  
Myocardial Cells ◽  
Expression Levels ◽  
Cardiac Functions

Abstract Objective ：The aim of this study was to determine whether Si-Miao-Yong-An decoction (SMYAD) could protect cardiomyocytes from ischemia/reperfusion (I/R) injury and its underlying mechanisms. Methods: C57BL/6 mice were used to establish a model of myocardial infarction by I/R injury and treated by SMYAD for 4 weeks. Then the cardiac functions of mice were evaluated by Cardiac Magnetic Resonance (CMR). Histopathological analysis for the heart remodeling was detected by H&E and Masson staining. The protein expression of Collagen I, MMP9 , and TNFα was detected by Western blot in the heart tissues. H9C2 cells were used to establish the hypoxia/reoxygenation (H/R) model and SMYAD intervention. MTT assays detected the cell viability of myocardial cells. The expression level of IL-1β was evaluated by ELISA. The expression levels of LC3B-II/LC3B‑I, p- mTOR, mTOR, NLRP3, pro-Caspase1, and cleaved-Caspase1 in H9C2 cells were evaluated by Western blot. Results : SMYAD improved cardiac functions such as ventricular volume and ejection fraction of the rats with ischemia/reperfusion injury. Morphological assay indicated that SMYAD reduced the scar size and inhibited fibrosis formation. It was found that SMYAD could regulate Collagen I, MMP9, and TNFα protein expression levels in the heart tissues. SMYAD improved the survival rate of cardiomyocytes H92C in the H/R injury model. SMYAD elevated the rate of LC3B-II/LC3B‑I protein expression, decreased the rate of p-mTOR/mTOR protein expression, and reduced expressions of Caspase 1, NLRP3, and IL-1β in H/R cardiomyocytes. Conclusion： SMYAD exerted protective effects on ischemia-reperfusion injury in myocardial cells by activating autophagy and inhibiting pyroptosis. This might be the reason why SMYAD protected myocardial tissue and improved cardiac function in mice with ischemia/reperfusion.

scholarly journals A Cell Model for Conditional Profiling of Androgen-Receptor-Interacting Proteins

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
K. A. Mooslehner ◽  
J. D. Davies ◽  
I. A. Hughes
Keyword(s):  
Androgen Receptor ◽  
Protein Interactions ◽  
Cell Model ◽  
Mouse Cell ◽  
Interacting Proteins ◽  
Wild Type ◽  
Genital Development ◽  
A Cell ◽  
The Impact ◽  
Male Genital

Partial androgen insensitivity syndrome (PAIS) is associated with impaired male genital development and can be transmitted through mutations in the androgen receptor (AR). The aim of this study is to develop a cell model suitable for studying the impact AR mutations might have on AR interacting proteins. For this purpose, male genital development relevant mouse cell lines were genetically modified to express a tagged version of wild-type AR, allowing copurification of multiprotein complexes under native conditions followed by mass spectrometry. We report 57 known wild-type AR-interacting proteins identified in cells grown under proliferating and 65 under nonproliferating conditions. Of those, 47 were common to both samples suggesting different AR protein complex components in proliferating and proliferation-inhibited cells from the mouse proximal caput epididymus. These preliminary results now allow future studies to focus on replacing wild-type AR with mutant AR to uncover differences in protein interactions caused by AR mutations involved in PAIS.

scholarly journals Tacrolimus reverses the pemphigus vulgaris serum-enhanced expression of desmoglein in HaCaT cells

2019 ◽  
Author(s):  
zhimin xie ◽  
Qiaolin Pan ◽  
Xucheng Shen ◽  
Yi Zhang ◽  
Xiangnong Dai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Cell Model ◽  
Pemphigus Vulgaris ◽  
Hacat Cells ◽  
Hacat Keratinocytes ◽  
Mrna Transcription ◽  
Rt Pcr ◽  
Enhanced Expression ◽  
A Cell

Abstract Pemphigus vulgaris (PV) is associated with autoantibodies against desmoglein (Dsg), including Dsg1 and Dsg3. However, the precise mechanism by which acantholysis occurs in response to PV-IgG and the effect of tacrolimus for PV remain unclear.Method To co-culture human HaCaT keratinocytes with DMEM medium containing 5% PV-sera to establish a cell model of pemphigus that can determine the effect of PV-sera and tacrolimus on Dsg mRNA transcription and protein expression in HaCaT cells. Dsg protein expression in HaCaT cells was evaluated by Western blotting and Dsg mRNA transcription by real-time PCR (RT-PCR). The distribution of Dsg1 and Dsg3 in HaCaT cells was determined by indirect immunofluorescence (IIF).Results The application of 5% PV serum resulted in an increase in the transcription and expression levels of Dsg1 and Dsg3, whereas tacrolimus suppressed Dsg1 and Dsg3 expression. Tacrolimus inhibited PV serum-induced disruption of cell−cell contacts. Tacrolimus also downregulated the expression of Dsg1and Dsg3 compared with the PV group. IIF revealed that the linear deposits of Dsg1 on the surface of HaCaT cells in the PV-sera group disappeared and were replaced by granular and agglomerated fluorescent particles on the cell surface, whereas the Dsg3 linear deposits still existed, but this effect could be reversed by tacrolimus.Conclusion The Dsg3 antibody disrupts desmosome junctions by inducing endocytosis, resulting in desmosomal dissociation. Tacrolimus could reverse PV serum-induced enhancement Dsg expression in HaCaT cells.

scholarly journals Si-Miao-Yong-An Decoction Maintains the Cardiac Function and Protects Cardiomyocytes from Myocardial Ischemia and Reperfusion Injury

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Wenwen Cui ◽  
Shen Xin ◽  
Lingjuan Zhu ◽  
Mingye Wang ◽  
Yuanyuan Hao ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Protein Expression ◽  
Western Blot ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
H9c2 Cells ◽  
Myocardial Cells ◽  
Expression Levels ◽  
Cardiac Functions

Objective. The aim of this study was to determine whether Si-Miao-Yong-An decoction (SMYAD) could protect cardiomyocytes from ischemia/reperfusion (I/R) injury and its underlying mechanisms. Methods. C57BL/6 mice were used to establish a model of myocardial infarction by I/R injury and treated by SMYAD for 4 weeks. Then, the cardiac functions of mice were evaluated by cardiac magnetic resonance (CMR). Histopathological analysis for the heart remodeling was detected by H&E and Masson staining. The protein expression of collagen I, MMP9, and TNFα was detected by western blot in the heart tissues. H9C2 cells were used to establish the hypoxia/reoxygenation (H/R) model and SMYAD intervention. MTT assays detected the cell viability of myocardial cells. The expression level of IL-1β was evaluated by ELISA. The expression levels of LC3B-II/LC3B-I, p-mTOR, mTOR, NLRP3, procaspase 1, and cleaved-caspase 1 in H9C2 cells were evaluated by Western blot. Results. SMYAD improved cardiac functions such as ventricular volume and ejection fraction of the rats with ischemia/reperfusion injury. Morphological assay indicated that SMYAD reduced the scar size and inhibited fibrosis formation. It was found that SMYAD could regulate collagen I, MMP9, and TNFα protein expression levels in the heart tissues. SMYAD improved the survival rate of H9C2 cardiomyocytes in the H/R injury model. SMYAD elevated the rate of LC3B-II/LC3B-I protein expression, decreased the rate of p-mTOR/mTOR protein expression, and reduced expressions of caspase 1, NLRP3, and IL-1β in H/R cardiomyocytes. Conclusion. SMYAD exerted protective effects on ischemia/reperfusion injury in myocardial cells by activating autophagy and inhibiting pyroptosis. This might be the reason why SMYAD protected myocardial tissue and improved cardiac function in mice with ischemia/reperfusion.

scholarly journals Tacrolimus reverses the pemphigus vulgaris serum-enhanced expression of desmoglein in HaCaT cells

2020 ◽  
Author(s):  
Zhimin Xie ◽  
Qiaolin Pan ◽  
Xucheng Shen ◽  
Yi Zhang ◽  
Xiangnong Dai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Cell Model ◽  
Pemphigus Vulgaris ◽  
Hacat Cells ◽  
Hacat Keratinocytes ◽  
Mrna Transcription ◽  
Rt Pcr ◽  
Enhanced Expression ◽  
A Cell

Abstract Background: Pemphigus vulgaris (PV) is associated with autoantibodies against desmoglein (Dsg), including Dsg1 and Dsg3. However, the precise mechanism by which acantholysis occurs in response to PV-IgG and the effect of tacrolimus on PV remains unclear. Method: Human HaCaT keratinocytes were co-cultured with DMEM medium containing 5% PV-sera to establish a cell model of pemphigus in order to determine the effect of PV-sera and tacrolimus on Dsg mRNA transcription and protein expression in HaCaT cells. Dsg protein expression in HaCaT cells was evaluated by Western blotting and Dsg mRNA transcription by real-time PCR (RT-PCR ). The distribution of Dsg1 and Dsg3 in HaCaT cells was determined by indirect immunofluorescence (IIF). Results: The application of 5% PV serum resulted in an increase in Dsg1 and Dsg3 transcription and expression levels, whereas tacrolimus suppressed Dsg1 and Dsg3 expression. Tacrolimus inhibited PV serum-induced disruption of cell-cell contacts. Tacrolimus also down-regulated Dsg1 and Dsg3 expression compared with PV. IIF revealed that Dsg1 linear deposits on the surface of HaCaT cells in the PV-sera group disappeared and were replaced by granular and agglomerated fluorescent particles on the cell surface, whereas the Dsg3 linear deposits were still present, however this effect could be reversed by tacrolimus. Conclusion: The Dsg3 antibody disrupts desmosome junctions by inducing endocytosis, resulting in desmosomal dissociation. Tacrolimus can reverse PV serum-induced enhancement Dsg expression in HaCaT cells.

Molecular Mechanisms of EML in AML1/ETO+ AML and Its Targeting Treatments

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5953-5953
Author(s):  
Fan Yi Meng ◽  
Ling Jiang ◽  
Qingxiu Zhong ◽  
Li Chun Wang ◽  
Guopan Yu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
In Vitro Study ◽  
Control Group ◽  
A Cell ◽  
App Gene

Abstract Our previous study has been reported that AML1/ETO positive patients with highly expressed of APP were much easier to extramedullary infiltration, and got poor prognosis£¨followed-up median 35 ( 6-96 ) months, RFS in the high expression APP group was significantly lower than the low expression group £¬40.0% vs 80.0%£¬P = 0.001). In vitro study, we constructed a cell model kasumi-1 which was consistent with AML1/ETO positive and high expressed of APP gene. The cell migration was significantly reduced after interferce of APP expression. This study was designed to investigate the molecule mechanism of extramedullary leukemia (EML) in kasumi-1 cell model and to invent a strategy for treatment in clinic. In this study, we found p-ERK, c-Myc and MMP-2 were significantly decreased after APP expression knockdown in kasumi-1 cell. Meanwhile, we added the inhibitors to block p-ERK and c-Myc expression. The results showed that protein expression of p-ERK and c-Myc was significantly decreased after p-ERK inhibitor performance, which was proportional to the time and concentration. c-Myc and MMP-2 protein expression was significantly reduced after c-Myc inhibitor was used, but p-ERK didn't change (Fig.1B). So, we concluded that APP might regulated the AML cell migration via APP/p-ERK/c-Myc/MMP-2 pathway. Also, we found that kasumi-1 cell was resistant to adriamycin (ADM) and Ara-C, which meant APP may be related with drug resistance. So, we detected cell surviving fraction after ADM and Ara-C performance via MTT assay. The results showed that there was no difference in control group and siAPP group. But, when compared with controls groups, cell surviving fraction in siEZH2 group was significantly decreased after ADM and Ara-C performance respectively. Furthermore, we found protein expression of EZH2 was significantly reduced after LBH589 treatment in cell culture. So, we concluded that LBH589 or SiEZH2 could increase sensitivity of kasumi-1 cell to ADM and Ara-C. In sum, in AML1/ETO positive leukemia cells, we first report that APP gene regulates cell migration via APP/p-ERK/c-Myc/MMP-2 pathway and EZH2 gene induces drug resistantence. Interference or blocking of EZH2 and APP expression may be helpful in treating AML1/ETO positive leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lipocalin-2 Mediated Polarization of Neutrophil in Cerebral Ischemia-Reperfusion Injury

2021 ◽  
Author(s):  
Zhiliang Guo ◽  
Guoli Xu ◽  
Jiaping Xu ◽  
Yaqian Huang ◽  
Chunfeng Liu ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cerebral Ischemia ◽  
Western Blot ◽  
Blood Brain Barrier ◽  
Cell Model ◽  
Ischemia Reperfusion ◽  
Inflammatory Factor ◽  
Lipocalin 2 ◽  
Cerebral Ischemia Reperfusion ◽  
A Cell

Abstract Cerebral ischemia-reperfusion (I/R) injury is a difficult point in the treatment of ischemic stroke. Lipocalin-2 (LCN2) and neutrophils play an important role in I/R injury. We explored the effect of anti-LCN2 antibody (LCN2mAb) and to further clarify the relationship between LCN2mAb and neutrophil polarization (N1/N2 neutrophils) in I/R injury. A mouse middle cerebral artery occlusion (MCAO) model was used to induce transient cerebral ischemia. LCN2mAb was administered 1h before MCAO; Anti-Ly6G was administered for 3d before MCAO. The expression of LCN2 and Ly6G was Measured by western blot. Infarct size,behavior and the blood-brain barrier (BBB) damage were assessed. The polarized N2 neutrophils were measured by western blot and Flow Cytometry. Using HL-60 as a cell model explores the role of LCN2mAb in the polarity transition of neutrophils. The expression of LCN2 and Ly6G in the brain at different time points reached a peak after I/R 24h and then gradually decreased. As demonstrated previously, LCN2mAb-treated mice had neuroprotective effects in cerebral infarction volume, behavior, and blood-brain barrier damage. The expression of Ly6G was not significantly different, but the expression of N2 neutrophils was increased. The expression of anti-inflammatory factor CD206 was significantly increased, and pro-inflammatory factor TNF-α was significantly reduced. Compared with mice treated with Anti-Ly6G, Anti-Ly6G-LCN2mAb combined treatment of I/R, there was no further improvement in behavior and pathology. Based on HL-60 as a cell model, N1-HL-60 cells were pretreated with LCN2mAb, and N2-HL-60 cells were significantly increased. LCN2 may affect the prognosis of ischemic stroke by mediating neutrophils polarization.

scholarly journals Tetrandrine Ameliorates Myocardial Ischemia Reperfusion Injury through miR-202-5p/TRPV2

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Wei Zhao ◽  
Youyang Wu ◽  
Fanhao Ye ◽  
Shiwei Huang ◽  
Hao Chen ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Myocardial Ischemia ◽  
Protein Expression ◽  
Western Blot ◽  
Ischemia Reperfusion ◽  
Cardiomyocyte Apoptosis ◽  
H9c2 Cells ◽  
Group I ◽  
Myocardial Ischemia Reperfusion ◽  
Myocardial Infarction Size

Objective. This study is aimed at investigating the therapeutic effects of tetrandrine (Tet) on myocardial ischemia reperfusion (I/R) injury and probe into underlying molecular mechanism. Methods. H9C2 cells were divided into hypoxia/oxygenation (H/R) group, H/R+Tet group, H/R+Tet+negative control (NC) group, and H/R+Tet+miR-202-5p inhibitor group. RT-qPCR was utilized to monitor miR-202-5p and TRPV2 expression, and TRPV2 protein expression was detected via western blot and immunohistochemistry in H9C2 cells. Cardiomyocyte apoptosis was evaluated through detection of apoptosis-related markers and flow cytometry. Furthermore, myocardial enzyme levels were detected by ELISA. Rats were randomly separated into sham operation group, I/R group, I/R+Tet group (50 mg/kg), I/R+Tet+NC group, and I/R+Tet+miR-202-5p inhibitor group. miR-202-5p and TRPV2 mRNA expression was assessed by RT-qPCR. TRPV2 protein expression was detected through western blot and immunohistochemistry in myocardial tissues. Apoptotic levels were assessed via apoptosis-related proteins and TUNEL. Pathological changes were observed by H&E staining. Myocardial infarction size was examined by Evans blue-TCC staining. Results. Abnormally expressed miR-202-5p as well as TRPV2 was found in H/R H9C2 cells and myocardial tissues of I/R rats, which was ameliorated following Tet treatment. Tet treatment significantly suppressed H/R- or I/R-induced cardiomyocyte apoptosis. ELISA results showed that CK-MB and LDH levels were lowered by Tet treatment in H/R H9C2 cells and serum of I/R rats. H&E staining indicated that Tet reduced myocardial injury in I/R rats. Also, myocardial infarction size was lowered by Tet treatment. The treatment effects of Tet were altered following cotreatment with miR-202-5p inhibitor. Conclusion. Our findings revealed that Tet may ameliorate myocardial I/R damage via targeting the miR-202-5p/TRPV2 axis.

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