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Methods The model of heart failure after myocardial infarction was established by left coronary artery liga-tion in rats. Two weeks after modeling, all rats were randomly divided into model group, LGZGD group, and captopril group. Meanwhile sham operation group was set up. The rats were given continuous intragastric administration with drug or distilled water for 28 days, once a day. The behavioral signs of rats in each group were observed. The cardiac function of rats in each group was examined by echocardiography. Serum BNP and NT-ProBNP content were detected by enzyme-linked immunoassay; The changes of myocardial his-topathological and collagen fibers in rats were detected using sirius staining. The contents of oxidative stress index including ROS, SOD in myocardial tissue of rats in each group were observed by DCFH-DA fluorescent probe and Enzyme-linked immunoassay. The ultra-structure of mitochondria was observed by transmission electron microscopy. Expressions of apoptotic proteins ( mitochondrial CytC, cytoplasmic CytC) were detec- ted by Western blot. Expression of proteins related to the Nrf2/BNIP3 pathway were examined by immunoflu-orescence and Western blot. Results LGZGD could significantly improve the cardiac function of rats, reduce the contents of BNP and NT-ProBNP, inhibit the excessive deposition of collagen in myocardial interstiti-um, reduce ROS, increase the content of SOD, improve mitochondrial structure damage, up-regulate the expression of Nrf2 and nuclear translocation, and reduce the expression of BNIP3. Conclusions LGZGD can inhibit the ventricular remodeling and prevent the occurrence of heart failure after myocardial infarction. Its pharmacological effects are mainly related to regulating the Nrf2/BNIP3 pathway, activating Nrf2, promoting its nuclear transfer, and further down-regulating BNIP3 , protecting mitochondrial function, and reducing cardiomyocyte apoptosis.
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Aim To investigate whether Linggui Zhugan Decoction ( LGZGD) can inhibit ventricular remodeling and prevent heart failure in rats after myocardial infarction by regulating Nrf2/BNIP3 pathway. Methods The model of heart failure after myocardial infarction was established by left coronary artery ligation in rats. Two weeks after modeling, all rats were randomly divided into model group, LGZGD group, and captopril group. Meanwhile sham operation group was set up. The rats were given continuous intragastric administration with drug or distilled water for 28 days, once a day. The behavioral signs of rats in each group were observed. The cardiac function of rats in each group was examined by echocardiography. Serum BNP and NT-ProBNP content were detected by enzyme -linked immunoassay; The changes of myocardial his-topathological and collagen fibers in rats were detected using sirius staining. The contents of oxidative stress index including ROS, SOD in myocardial tissue of rats in each group were observed by DCFH-DA fluorescent probe and Enzyme-linked immunoassay. The ultra-structure of mitochondria was observed by transmission electron microscopy. Expressions of apoptotic proteins ( mitochondrial CytC, cytoplasmic CytC) were detected by Western blot. Expression of proteins related to the Nrf2/BNIP3 pathway were examined by immunofluorescence and Western blot. Results LGZGD could significantly improve the cardiac function of rats, reduce the contents of BNP and NT-ProBNP, inhibit the excessive deposition of collagen in myocardial interstiti-um, reduce ROS, increase the content of SOD, improve mitochondrial structure damage, up-regulate the expression of Nrf2 and nuclear translocation, and reduce the expression of BNIP3. Conclusions LGZGD can inhibit the ventricular remodeling and prevent the occurrence of heart failure after myocardial infarction. Its pharmacological effects are mainly related to regulating the Nrf2/BNIP3 pathway, activating Nrf2, promoting its nuclear transfer, and further down-regulating BNIP3, protecting mitochondrial function, and reducing cardiomyocyte apoptosis.
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BACKGROUND: Different intensities of exercises make different effects on human body, and the changes of skeletal muscle after exercise remain unclear. Physical change of human body during exercise Is a hotspot. OBJECTIVE: To Investigate the effect of different Intensities of exercises on the mass of rat skeletal muscle, and the role of BNIP3-mediated mitophagy In maintaining skeletal muscle mass. METHODS: The study was approved by the Laboratory Animal Ethical Committee of Beijing Sport University. Twenty-four healthy male Sprague-Dawley rats aged 8 weeks were randomly divided Into three groups: Control, moderate-intenslty exercise (5°, 15 m/min, 1 hour, 60% VOanux) and hlgh-lntenslty exercise (5°, 35 m/min, 20 minutes, 85% V02max) groups (n=8/group), 6 times weekly. The rat soleus and gastrocnemius were removed to measure the wet weights. The cross-sectional area of muscle fiber was detected by immunofluorescence. The protein expression levels of BNIP3, p62 and LC3 in the soleus and gastrocnemius were detected by western blot assay. RESULTS AND CONCLUSION: (1) The wet weight of gastrocnemius in the exercise groups was significantly lower than that in the control group (P < 0.01). (2) The cross-sectional area of gastrocnemius fiber in the exercise groups was significantly less than that in the control group (P < 0.01), and the cross-sectional area of soleus fiber in the exercise groups was significantly more than that in the control group (P < 0.01). (3) The moderate-intensity exercise induced increased mitophagy, and the expression level of BNIP3 and LC3-II/LC3-I were up-regulated (P < 0.05), while p62 was down-regulated (P < 0.05) compared with the control group. The expression level of LC3-II/LC3-I in the high-intensity exercise group was higher (P < 0.05), but the expression level of p62 was lower than that in the moderate-intensity exercise group and the expression of BNIP3 was decreased (P < 0.05). (4) To conclude, 4-week moderate-intensity exercise can promote the removal of damaged mitochondria and maintain skeletal muscle function by increasing BNIP3-regulated mitophagy in skeletal muscle. In high-intensity exercise, the level of autophagy is higher, but will cause harmful effect on skeletal muscle.
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Objective:To study the regulatory level of Dima decoction on adenovirus E1B 19 kD interacting protein 3（BNIP3） protein and to understand the mechanism in intervening mitophagy and controlling inflammation in ulcerative colitis. Method:The 60 SPF SD rats were randomly divided into normal control group, model control group, low, medium ang high dose Dima decoction（8.5，17.0，34.0 g·kg-1）group and mesalazine（3.0 g·kg-1） group，10 for each group. The model of UC rats was established by the method of environmental diet intervention +2,4,6-trinitro-Benzenesulfonicacid（TNBS）+ethanol，the administration group was administered for 14 days.Hematoxylin-eosin (HE) was used to detect pathological changes of colonic mucosal tissues in rats.Western blot and real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） were used to detect the expressions of BNIP3 protein and mRNA in intestinal epithelial cells. Result:HE staining results showed that a large number of inflammatory cell infiltration and non-cheesy granulation tissue were observed in the colon of rats in the model group, while the rats in each drug group showed different degrees of fibrous repair. Compared with normal group, the expression of BNIP3 protein and mRNA in model group were significantly increased (P<0.05). Compared with model group, the expression of BNIP3 protein and mRNA in low, medium and high-dose Dima decoction group and mesalazine group were significantly increased (P<0.01). Compared with low-dose group, the expression of BNIP3 protein and mRNA in medium and high-dose groups and mesalazine group were significantly increased (P<0.05). Conclusion:Dima decoction can increase the expression of BNIP3 in the intestinal epithelium of UC active rats, which may be one of the mechanisms of promoting mitochondrial autophagy against UC inflammation.
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Objective To study the regulating mechanisms of cobaltous chloride (CoCl2)induced hypoxia on muscle atrophy.Methods The mouse skeletal C2C12 myotube line was used as the cell model and divided into a control group,a CoCl2 group,a 3-Methyladenine(3MA)group and a CoCl2+3MA group.The control group was not given any treatment,the CoCl2 group was treated with 200 μM CoCl2 to induce hypoxia,and the 3MA group or CoCl2+3MA group was treated with 5 mM 3MA in the absence or presence of 200 μM CoCl2.The Giemsa stain was conducted to observe the morphology of myotubes.The multifunctional microplate reader was utilized to quantify the reactive oxygen species (ROS)expression.The autophagosome was observed by using the transmission electron microscopy.The mRNA and protein expression of hypoxia-inducible factor-1α(HIF-1α),Bcl2/adenovirus E1B19kDa interacting protein 3(BNIP3),microtubule associated protein1 light chain 3(LC3)were detected using the quantitative real time polymerase chain reaction (QRT-PCR)and Western blotting (WB).Moreover,3-Methyladenine(3MA)was used to suppress autophagy and the expression of muscle atrophy F-box(MAF-bx)was detected.Results More spindly ring-shaped myotubes were formed in the control group,while CoCl2 induced myotubes atrophy and rupture.The expression of ROS increased significantly in the CoCl2 group compared with the control group(t=-4.965,P=0.008).Transmission electron microscopy results showed autophagosome formation induced by CoCl2,and significant improvement in the HIF-1α,BNIP3 and LC3 in the CoCl2 group compared with the control group(P＜0.05).The protein expression of MAFbx decreased when co-cultured with CoCl2 and 3MA(F=18.246,P=0.001).Conclusions The skeletal muscle atrophy caused by CoCl2 induced hypoxia may be associated with autophagy promoted by the HIF-1α/BNIP3 signal pathway,and inhibition of hypoxia induced autophagy can partly reduce the atrophy in skeletal C2C12 myotubes.
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Objective To evaluate the effect of autophagy-related factors LC3 and Bnip-3 and apoptosis related factors Bcl-2 and Bax on brain damage in experimental diabetic rats .Methods Thirty SD rats were randomly divided into diabetic group and control group .The diabetic group was injected with 1% streptozotocin ( 60 mg/kg body mass ) and the control group with citrate buffer .The rats were sacrificed after 12 weeks feeding and brain tissues were obtained .Pathological chan-ges were observed and the expression of LC 3, Bnip-3, Bcl-2 and Bax in brain tissues of the rats was detected by immuno-histochemical SP method .Results Compared with the control group ,the diabetic rat brain pathology showed that the cell arrangment was more disorderly and distributed more unevenly , the cell body was smaller , cytoplasm was lighter red , and the number of nerve cells of normal morphology was smaller .The positive number of LC3, Bnip-3 and Bax in brain tissues of diabetic rats was significantly larger than in the control group ,and the difference was statistically significant (P<0.05). However ,the positive number of Bcl-2 was significantly smaller than in the control group ,and the difference was statistically significant (P<0.001).In diabetic rats, LC3 and Bnip-3 showed a weak positive correlation(P<0.05), Bcl-2 and Bax were irrelevant, Bnip-3 and Bax were positively weakly-correlated(P<0.05),Bcl-2 was not correlated;and Bcl-2 and Bax were irrelevant.Conclusion LC3,Bnip-3 and Bax in the brain tissue of diabetic rats are overexpressed , while Bcl-2 shows weak expression , indicating that autophagy factors and apoptotic factors are involved in the process of brain injury in diabet -ic rats, which may be one of the mechanisms of brain tissue damage .
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<p><b>OBJECTIVE</b>This study investigated the expression of BNIP3 in salivary adenoid cystic carcinoma (SACC) and its correlations to the clinicopathological features and prognosis of patients with SACC. The role of BNIP3 in the progress of hypoxia-induced autophagy was elucidated.</p><p><b>METHODS</b>The expression levels of BNIP3, hypoxia inducible factor (HIF)-1α, and LC3 in 65 SACC cases were detected by immunohistochemical staining method, and the correlation between the expression of BNIP3 and the clinicopathological features in SACC was analyzed. In addition, the correlations of BNIP3 gene expression with HIF-1α and LC3 gene expression were analyzed. The survival rate of patients with SACC was evaluated by univa-riate survival analysis.</p><p><b>RESULTS</b>BNIP3 was considerably expressed in SACC in all three histological patterns, and was positive in 41 cases (63.1%). BNIP3 gene expression was significantly correlated with histological grade (P=0.001) and HIF-1α gene expression (P=0.011). By contrast, BNIP3 gene expression was not significantly correlated with LC3 gene expression (P= 0.167). The overall survival rate of patients with negative BNIP3 expression was better than that of patients with positive BNIP3 expression (P<0.05).</p><p><b>CONCLUSIONS</b>BNIP3 might play a vital role in the tumorigenesis of SACC and may be a new target for gene therapy. .</p>
Subject(s)
Humans , Autophagy , Carcinoma, Adenoid Cystic , Contraindications , Hypoxia-Inducible Factor 1, alpha Subunit , Membrane Proteins , Prognosis , Proto-Oncogene Proteins , Salivary Gland NeoplasmsABSTRACT
Objective To detect the methylation status of the promoter of BNIP3 gene in gastric cancer cell lines MKN1,and to explore the mecha?nism of DNA methylation regulating the expression of BNIP3 in gastric cancer cells. Methods The methylation status of BNIP3 promoter was de?tected by bisulfate sequencing PCR. Reverse transcription PCR was used to evaluate BNIP3 mRNA expression. MKN1 cells were treated with 5?Aza?2′?deoxycytidine(5?Aza?CdR),and after the treatment,the methylation status and BNIP3 mRNA expression were observed. Chromatin immuno?precipitation(ChIP)was used to determine the combination of BNIP3 with DNA methyltransferase 1(DNMT1). Results The promoter DNA of BNIP3 in MKN1 cells was in state of hypermethylation. Compared to the control group,methylation status and mRNA expression of BNIP3 in the drug treatment group(the 5?Aza?CdR concentration was 10μmol/L)were reversed,which showed statistical differences(P<0.05). 5?Aza?CdR inhibited the combination of BNIP3 with DNMT1. Conclusion CpG island methylation regulates BNIP3 gene expression in MKN1 cells. DNA methylation is related with the binding between the promoter of BNIP3 and DNMT1.
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[Abstract ] Objective The purpose of this study was to observe the effects of dl-3n-butylphthalide (NBP) sodium chloride injection post-processing on the expressions of X-inhibitor of apoptosis (XIAP) and Bcl-2/adenovirus E1B19kDa interacting protein 3 (BNIP3) in the hippocampus CA1 neurons of focal cerebral ischemia reperfusion (IR) rats, and to investigate the brain-protection mechanisms of NBP. Methods A total of65 adult male Sprague-Dawley rats were divided into five groups of equal number, sham op-eration, IR, and low-,medium -and high-dose NBP, according to the random number table. The IR models were established by modified ligation of the middle cerebral artery.The animals in the NBP groups received intra-abdominal injection of NBP at 2, 4, and 6 mg/kg, re-spectively.All the rats were sacrificed at 24 hours after modeling,neurological scores obtained by Zea Longa, the volume of infarction measured by TTC staining, the number of apoptotic cells counted by TUNEL, and the expressions of XIAP and BNIP3 detected by immunohistochemistry and real-time PCR. Results The neural function defect scores were markedly lower in low-, medium-and high-dose NBP groups than in IR model rats (P<0.05), with statis-tically significant differences among the three dose groups (P<0.05).The volume of infarction was remarkably higher in the low-dose than in the medium-and high-dose NBP groups (P<0.05).The number of apoptotic cells in the hippocampus CA1 neurons was de-creased in the NBP groups as compared with the IR models (P<0.05).The XIAP-and BNIP3-positive cells were significantly in-creased in the IR model rats as compared with the sham operation group ([22.31 ±0.94] and [60.13 ±2.59]/HP vs [3.07 ±1.43] and [5.78 ±0.44]/HP, P<0.05).In comparison with the IR models, the NBP-treated rats showed a progressively increased number of XIAP-positive cells in low-, medium-, and high-dose groups ([28.70 ±1.18], [32.79 ±0.88], and [37.01 ±1.24]/HP) (P<0.05) but a decreased number of BNIP3-positive cells in the three dose groups ([52.07 ±1.02], [40.30 ±2.00], and [31.04 ± 0.43]/HP) (P<0.05).Similarly, the expression of XIAP mRNA was up-regulated while that of BNIP3 mRNA down-regulated in the NBP treatment groups as compared with the IR model rats, both in a dose-dependent manner (P<0.05). Conclusion NBP post-processing has a neuroprotective effect on IR rats, which is associated with its impact on the expressions of XIAP and BNIP3.
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Mitochondrial quality control is the important mecha-nism that regulates the morphology,quantity and quality of mito-chondrial in cell to maintain cellular homeostasis and thus,cell survival and health.It has been revealed that members of Bcl-2 family are linked to mitochondrial function and integrity.Bcl-2 family proteins are the key regulators of mitochondrial quality control,participating in the signaling pathways regulating the crosstalk between mitophagy and apoptosis,as well as mitochon-drial fission and fusion.This paper mainly reviews their impact on mitochondrial quality and the major signaling pathways regula-ted by Bcl-2 family proteins.
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Aim To study the effect of BNIP3 on hy-poxia-induced autophagic cell death in gastric carcino-ma.Methods The protein levels of BNIP3 and LC3-Ⅱ of 1 8 cases of gastric carcinoma were studied with immunocytochemistry and Western blot analysis be-tween normal and tumor tissues.The expression of LC3-Ⅱ in SGC-7901 cells was assessed while knock-down of BNIP3 by siRNA in CoCl2-induced hypoxia, and the effect of 3-MA was also studied.Cell viability in hypoxia treatment for 48 h was estimated with cell counting kit-8 (CCK8).Results Compared with nor-mal tissues,the protein levels of BNIP3 and LC3-Ⅱwere increased in tumor tissues.BNIP3 siRNA could decrease the expression of LC3-Ⅱ and enhance the cell viability,while inhibition of autophagy could also in-crease the cell viability in SGC-7901 cells.Conclu-sions BNIP3 may play a role in hypoxia-induced cell death in gastric carcinoma,and the possible mecha-nism may be related to the regulation of autophagy.
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Bnip3,a kind of mitochondrion apoptosis gene,belongs to the Bcl-2 gene family and BH3-only subfamily.It is one of the downstream response factors of hypoxia inducible factor (HIF).Bnip3 can induce cell apoptosis and autophagy under the oxygen deficit condition.Many studies show that Bnip3 is closely related to the occurrence,development and treatment of tumors.Molecular targeting treatment aimed at Bnip3 combined with chemoradiotherapy has preferable application foreground in tumor therapy.
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Apoptosis disorders have an important function in the development of gastrointestinal cancer. BNIP3 is a member of the BH3-only subfamily of the bcl-2 family, which contains a BH3 domain and a transmembrane domain, and belongs to the mitochon-drial pro-apoptotic proteins. BNIP3 induces cell death via the caspase-independent mitochondrial apoptotic pathway and mediates au-tophagic cell death. BNIP3 expression is regulated by hypoxia and other factors. BNIP3 expression in tumors exhibits tissue specificity;BNIP3 is highly expressed in some tumors, including breast, lung, and cervical tumors. In pancreatic, gastric, and colorectal cancers, BNIP3 is epigenetically silenced. The absence of BNIP3 in the tumors can cause tumor cells to tolerate hypoxia and may be associated with chemotherapy and radiotherapy resistance. A comprehensive understanding of the spectrum of BNIP3 expression in a various tu-mors is necessary to use BNIP3 as a marker in clinical applications to treat tumors and as a new target in tumor prognosis.
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PURPOSE : Cell death deregulation is a hallmark of human cancers. BNIP3, which was initially identified as a pro-apoptotic member of the Bcl-2 family, plays an important role in apoptosis, necrosis and autophagy. This study was conducted to explore whether mutation of the BNIP3 gene is a characteristic of human non-small cell lung cancers (NSCLC). MATERIALS AND METHODS : In the current study, we used polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and DNA sequencing to detect somatic mutations in the DNA sequences encoding the BH3 (Bcl-2 homology3) and TM (transmembrane) domains that are important to the cell death function of BNIP3 in 48 NSCLCs. RESULTS : SSCP analysis revealed no evidence of somatic mutation in the DNA sequences encoding the BH3 and TM domains of the human BNIP3 gene in the 48 NSCLCs evaluated in this study. CONCLUSION : The data presented here indicate that the pro-apoptotic BNIP3 gene may not be somatically mutated in human NSCLCs, which suggests that mutational events of the BNIP3 gene may not be involved in the mechanisms by which NSCLCs evade cell death
Subject(s)
Humans , Apoptosis , Autophagy , Base Sequence , Carcinoma, Non-Small-Cell Lung , Cell Death , Lung Neoplasms , Lung , Necrosis , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNAABSTRACT
Apoptosis is regulated by interaction of antiapoptotic Bcl-2 family proteins with various proapoptotic proteins, several of which are also members of the Bcl-2 family. BNIP3 (formerly NIP3) is a proapoptotic mitochondrial protein classified in the Bcl-2 family based on limited sequence homology-3 (BH3) domain and COOH-terminal transmembrane domain. Sequence comparison of BNIP3 has indicated that there are several BNIP3 human homologs of this protein, like BNIP3L, Nix and BNIP3. We have cloned a new member of BNIP3 family from the cDNA library prepared from human dermal papilla cells and designated as BNIP3h. BNIP3h shows substantial homology with other BNIP3 family proteins. BNIP3h induced apoptosis from 24 hours after transfection in MCF7 cell lines and its apoptosis inducing activity is extended until 72 hours after transfection.
Subject(s)
Humans , Amino Acid Sequence , Apoptosis/physiology , Base Sequence , Cells, Cultured , Cloning, Molecular , Dermis/chemistry , Membrane Proteins/chemistry , Mitochondria/chemistry , Molecular Sequence Data , Multigene Family , Sequence Alignment , Tissue Distribution , Transfection , Tumor Cells, CulturedABSTRACT
Using the technique of fluorescent-labled mRNA differential display, new apoptosis related gene 2ass-bnip3 of type 2 diabetic cardiomyopathy was found, the sequence of 1594 bp with coding 187 amino acids was obtained by the full-length clone, and its structural and functional predictions were performed. 2ass-bnip3 may play an important role in the development of diabetic cardiomyopathy via a regulatory pathway of calcium regulation and apoptosis.