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1.
Article in Chinese | WPRIM | ID: wpr-940983

ABSTRACT

OBJECTIVE@#To explore whether the using of mimetic peptide Gap27, a selective inhibitor of connexin 43 (Cx43), could block the death of dopamine neurons and influence the expression of Cx43 in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease mouse models.@*METHODS@#Eighteen C57BL/6 mice were randomly divided into control group, 6-OHDA group and 6-OHDA+Gap27 group, with 6 mice in each group. Bilateral substantia nigra stereotactic injection was performed. The control group was injected with ascorbate solution, 6-OHDA group was injected with 6-OHDA solution, and 6-OHDA+Gap27 group was injected with 6-OHDA and Gap27 mixed solution. Immuno-histochemical staining was used to detect the number of dopamine neurons, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of Cx43 messenger ribonucleic acid (mRNA), immuno-fluorescence staining was used to detect the distribution of Cx43 protein, the contents of Cx43 protein and Cx43 phosphorylation at serine 368 (Cx43-ps368) in mouse midbrain were detected by Western blot.@*RESULTS@#After injection of 6-OHDA, numerous dopamine neurons in substantia nigra died as Cx43 content increased, Cx43-ps368 content decreased. Mixing Gap27 while injecting 6-OHDA could reduce the number of death dopamine neurons and weaken the changes of Cx43 and Cx43-ps368 content caused by 6-OHDA. The number of tyrosine hydroxylase (TH) immunoreactive positive neurons in 6-OHDA group decreased to 27.7% ± 0.02% of the control group (P < 0.01); The number of TH immunoreactive positive neurons in 6-OHDA+Gap27 group was (1.64±0.16) times higher than that in 6-OHDA group (P < 0.05); The content of total Cx43 protein in 6-OHDA group was (1.44±0.07) times higher than that in 6-OHDA+Gap27 group (P < 0.05) while (1.68±0.07) times higher than that in control group (P < 0.01). In 6-OHDA group, the content of Cx43-ps368 protein and its proportion in total Cx43 protein were significantly lower than that in 6-OHDA+Gap27 group (P < 0.05).@*CONCLUSION@#In 6-OHDA mouse models, mimetic peptide Gap27 played a protective role in reducing the damage to substantia nigra dopamine neurons, which was induced by 6-OHDA. The overexpression of Cx43 protein might have neurotoxicity to dopamine neuron. Meanwhile, decreasing Cx43 protein level and keeping Cx43-ps368 protein level may be the protective mechanisms of Gap27.


Subject(s)
Animals , Connexin 43/pharmacology , Disease Models, Animal , Dopaminergic Neurons/metabolism , Mice , Mice, Inbred C57BL , Oxidopamine/metabolism , Parkinson Disease/metabolism , Peptides/pharmacology , Tyrosine 3-Monooxygenase/pharmacology
2.
Acta Pharmaceutica Sinica B ; (6): 3063-3072, 2022.
Article in English | WPRIM | ID: wpr-939951

ABSTRACT

Adipose tissue is a promising target for treating obesity and metabolic diseases. However, pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization, especially in obese subjects. We have previously shown that during cold exposure, connexin43 (Cx43) gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells. We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue. Using an adipose tissue-specific Cx43 overexpression mouse model, we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of the β 3-adrenergic receptor agonist Mirabegron and FGF21. Additionally, combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy. In light of these findings, we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it. Thus, Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.

3.
Article in Chinese | WPRIM | ID: wpr-933298

ABSTRACT

Objective:To evaluate the effect of rat cardiac fibroblasts (RCF) on the expression of connexin43 (Cx43) in H9c2 cells during hypothermic hypoxia/reoxygenation.Methods:H9c2 cells cultured in vitro were divided into 4 groups ( n=12 each) using the random number table method: control group (group C), hypothermic hypoxia/reoxygenation group (group HHR), RCF co-culture group (group Co) and RCF co-culture plus hypothermic hypoxia/reoxygenation group (group Co+ HHR). Group C was incubated at 37℃ in 5% CO 2 + 95% air for 5 h. Group HHR was incubated at 4 ℃ in 5% CO 2 + 95% N 2 for 1 h and then at 37 ℃ in 5% CO 2 + 95% air for 4 h. In group Co and group Co+ HHR, H9c2 cells 0.3×10 5 cells/well were inoculated in the lower chamber and RCF 0.6×10 5 cells/well in the the upper chamber of a transwell ? culture dish.Group Co was incubated at 37 ℃ in 5% CO 2 + 95% air for 5 h. Group Co+ HHR was incubated at 4℃ in 95% N 2 + 5% CO 2 for 1 h, and then incubated at 37 ℃ in 5% CO 2 + 95% air for 4 h. The mortality rate of H9c2 cells was measured by trypan blue staining, the expression of Cx43 and extracellular signal-regulated protein kinases 1/2 (ERK1/2) by immunofluorescence, and the expression of Cx43, phosphorylated Cx43, ERK1/2 and phosphorylated ERK1/2 by Western blot. Results:Compared with group C, the mortality rate of H9c2 cells was significantly increased, the expression and phosphorylation of Cx43 were decreased, and the expression and phosphorylation of ERK1/2 were increased in group HHR ( P<0.05), and no significant change was found in the mortality rate of H9c2 cells or expression and phosphorylation of Cx43 and ERK1/2 in group Co ( P>0.05). Compared with group Co, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Compared with group HHR, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Conclusions:RCFs can decrease the expression and activity of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation, and the mechanism may be related to the down-regulation of ERK1/2 expression and inhibition of ERK1/2 activity.

4.
Chinese Journal of Orthopaedics ; (12): 1152-1162, 2021.
Article in Chinese | WPRIM | ID: wpr-910702

ABSTRACT

Objective:To investigate the expression of connexin-43 (Cx43) in steroid-induced osteonecrosis of femoral head and osteoblasts in rats and its regulation mechanism.Methods:The model of steroid-induced osteonecrosis of femoral head (SIONFH) of rat was established. Micro-CT and HE staining were used to observe the degree of bone trabecular destruction and the incidence of empty lacunae. The expression levels of Cx43 and PI3K/Akt signaling pathway related molecules and osteoblast-related proteins in model group and control group were detected by RT-PCT and Western blot. The osteoblast (OB) of rats was further isolated and cultured in vitro. Under treatment of dexamethasone (Dex), Cx43 expression in OB cells was detected by Western blot and immunofluorescence. Western blot was used to detect the effect of glucocorticoid (GC) on the expression of related molecules of PI3K/Akt/β-catenin signaling pathway. Akt activator (SC79) and PI3K inhibitor (LY294002) were used to study the molecular mechanism of Dex regulation on Cx43 expression in OB cells. The regulatory relationship between β-catenin and Cx43 was investigated by immunoprecipitation and small interfere RNA (siRNA) technology.Results:The model of SIONFH in rats was successfully established, which proved that Cx43 expression level in the SIONFH model group was significantly lower than that in the control group, and the expression level of Cx43 was positively correlated with the expression of PI3K/Akt signaling pathway related molecules and osteoblast-related proteins Runx2, ALP and Collagen I Type (COL). In addition, in vitro culture of isolated rat OB cells, the expression of Cx43, p-PI3K, P-Akt and β-catenin in OB cells decreased gradually as the Dex action time went on. Moreover, SC79 pretreatment could significantly reverse the inhibitory effect of GCs on Cx43 expression, while LY294002 could significantly enhance the inhibitory effect of GCs on Cx43. In addition, the immunoprecipitation results showed that β-catenin expression was closely related to Cx43 expression, and further studies showed that β-catenin-siRNA could significantly down-regulate the expression of Cx43.Conclusion:Under the action of GC, the expression level of Cx43 in bone tissue and OB cells decreased significantly, and the possible mechanism was that GCs inhibited the expression of Cx43 by inhibiting the PI3K/Akt/β-catenin signaling pathway, which laid a new theoretical foundation for the further study of the role of Cx43 in the pathogenesis of steroid-induced femoral head necrosis.

5.
Acupuncture Research ; (6): 269-274, 2020.
Article in Chinese | WPRIM | ID: wpr-844163

ABSTRACT

OBJECTIVE: To compare the therapeutic effect of shallow and deep needling at "Neiguan"(PC6) in the treatment of arrhythmia in rabbits. METHODS: Male New Zealand rabbits were randomly divided into saline (n=15), model (n=12), shallow needling (n=13) and deep needling (n=12) groups. The arrhythmia model was established by ear intravenous injection of Barium chloride (0.4%, 1 mL/kg). Acupuncture needle was inserted into the superficial fascia (about 3 mm beneath the skin) or deep layer (5 to 8 mm, near the median nerve) of local PC6 tissue (fore limb), manipulated for a while and then retained for 10 min. Histopathological changes of myocardium was observed after H.E. stain, and the immunoactivity of connexin 43 (Cx43) detected by immunohistochemistry(IHC). RESULTS: Various degrees of arrhythmia and down-regulated expression of myocardial Cx43 were observed in all rabbits after modeling. After EA intervention and compared with the model group, the initial time of arrhythmia and Cx43 expression were obviously increased (P<0.01), and the duration of arrhythmia was significantly shortened in both deep and shallow needling groups (P<0.01). Compared with the shallow needling group, the Cx43 expression was increased in the deep needling group (P<0.01). H.E. staining showed disordered and wavy arrangement of myocardial fibers, with exudation of serous and erythrocytes in the myocardial interstitium in the model group, which was relatively mild in both needling groups. IHC showed disordered distribution of Cx43 in the ventricular myocytes and almost no obvious band-like distribution at the discs in rabbits of the model group, and abundant distribution of Cx43 in the sacral disc in the deep needling group, and strip-shaped, cluster-like, point-like, visible at both end-to-end connections and side-to-side connections in the shallow needling group. CONCLUSION: Both shallow and deep needling can significantly reduce the duration of arrhythmia in arrhythmia rabbits, which may be associated with its effect in up-regulating expression of myocardial Cx43 protein.

6.
Article in Chinese | WPRIM | ID: wpr-862691

ABSTRACT

<b>Objective::To investigate the mechanism of Buyang Huanwu Tang (BYHWT) in improving synaptic structural plasticity after cerebral ischemia-reperfusion in rats. <b>Method::Middle cerebral artery occlusion and reperfusion model was established. SD rats were randomly divided into sham-operated group, model group, BYHWT group, BYHWT+ Gap26(connexin43 inhibitor)groups. BYHWT was given twice a day(16 g·kg<sup>-1</sup>), Gap26 was intraperitoneally injected once a day since the third day after surgery (25 g·kg<sup>-1</sup>). Brain was taken out at the 7<sup>th</sup> day. The changes of neuronal synaptic and gap junction ultrastructure were observed by transmission electron microscopy. Synaptophysin (SYN) and growth-associated protein-43 (GAP-43) protein expression were detected by Western blot and immunofluorescence. <b>Result::The structure of synapses was integrated, and the gap junctions were clear in sham-operated group. In the hippocampus of model group, the structure was destroyed, and the gap junctions disappeared. Compared with the sham-operated group, model group up-regulated the expressions of SYN and GAP-43 (<italic>P</italic><0.05, <italic>P</italic><0.01). In the hippocampus of BYHWT group, the structure was close to the normal. Furthermore, BYHWT up-regulated the expressions of SYN and GAP-43 (<italic>P</italic><0.05, <italic>P</italic><0.01). However, after the combined administration with Cx43 inhibitor (Gap26), the damage of synaptic structural decreased, only a small number of gap junctions with the structural integrity can be seen, and the effect of BYHWT on SYN and GAP-43 was inhibited (<italic>P</italic><0.05, <italic>P</italic><0.01). <b>Conclusion::BYHWT could improve the hippocampal synaptic structural plasticity obviously after the CIRI. The mechanism may be related to the increase of the expression of Cx43 and the promotion of the intervention of SYN and GAP-43.

7.
Article in Chinese | WPRIM | ID: wpr-847434

ABSTRACT

BACKGROUND: Connexin 43 (Cx43) plays an important role in occurrence and development of osteoarthritis. However, the specific mechanisms involved remain unclear. OBJECTIVE: To verify the possibility of the dominant position of Cx43 in connexin family in osteoarthritis by detecting the expression of Cx43 in articular cartilage and chondrocyte cell line, and to construct shRNA lentivirus vector of Cx43 gene and establish a stable transfer cell line of chondrocyte (SW1353). METHODS: Animal models of osteoarthritis were established in six C57BL/6 mice by anterior cruciate ligament transection. The differences of Cx43 expression between osteoarthritic and normal knees were investigated by immunohistochemistry. Expression of Cx43 mRNA in chondrocyte (SW1353) was detected by RT-PCR, and the expression levels of Cx37, Cx40, Cx45 and Cx46 in SW1353 cells were detected as control. Cx43 were connected to the lentiviral vector carrying the EGFP gene, to reconstruct the lentiviral vector plasmid. The viral particles were generated by co-transfection of 293T cells with Cx43-shRNA. After transfection of Cx43-shRNA lentiviral vector into chondrocytes (SW1353), the expression level of Cx43 was detected by western blot assay and RT-PCR. The study protocol was approved by the Ethics Committee of State Key Laboratory of Oral Diseases, approved No. SKLODLL2013A172. RESULTS AND CONCLUSION: The expression level of Cx43 was significantly increased in the articular cartilage of osteoarthritic knees. The expression level of Cx43 mRNA was significantly higher than that of Cx37, Cx40, Cx45 and Cx46 in chondrocytes (SW1353). In SW1353 cells, Cx43 occupied the dominant position in connexin family. Cx43 shRNA lentiviral vector could inhibit the expression of Cx43 mRNA in SW1353 cells. The stably transfected SW1353 cell line was screened, laying a foundation for verifying the role of Cx43 in osteoarthritis.

8.
Article in English | WPRIM | ID: wpr-787143

ABSTRACT

The present study was aimed to explore the neuroprotective role of imatinib in global ischemia-reperfusion-induced cerebral injury along with possible mechanisms. Global ischemia was induced in mice by bilateral carotid artery occlusion for 20 min, which was followed by reperfusion for 24 h by restoring the blood flow to the brain. The extent of cerebral injury was assessed after 24 h of global ischemia by measuring the locomotor activity (actophotometer test), motor coordination (inclined beam walking test), neurological severity score, learning and memory (object recognition test) and cerebral infarction (triphenyl tetrazolium chloride stain). Ischemia-reperfusion injury produced significant cerebral infarction, impaired the behavioral parameters and decreased the expression of connexin 43 and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the brain. A single dose administration of imatinib (20 and 40 mg/kg) attenuated ischemia-reperfusion-induced behavioral deficits and the extent of cerebral infarction along with the restoration of connexin 43 and p-STAT3 levels. However, administration of AG490, a selective Janus-activated kinase 2 (JAK2)/STAT3 inhibitor, abolished the neuroprotective actions of imatinib and decreased the expression of connexin 43 and p-STAT3. It is concluded that imatinib has the potential of attenuating global ischemia-reperfusion-induced cerebral injury, which may be possibly attributed to activation of JAK2/STAT3 signaling pathway along with the increase in the expression of connexin 43.


Subject(s)
Animals , Brain , Carotid Arteries , Cerebral Infarction , Connexin 43 , Imatinib Mesylate , Ischemia , Learning , Memory , Mice , Motor Activity , Neuroprotection , Phosphotransferases , Reperfusion , Reperfusion Injury , STAT3 Transcription Factor , Transducers , Walking
9.
Rev. bras. cir. cardiovasc ; 34(6): 711-722, Nov.-Dec. 2019. tab, graf
Article in English | LILACS | ID: biblio-1057503

ABSTRACT

Abstract Objective: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). Methods: The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. Results: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. Conclusion: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Young Adult , Atrial Fibrillation/metabolism , Cytoskeleton/metabolism , Nuclear Proteins/metabolism , Connexin 43/metabolism , Myocytes, Cardiac/cytology , Adaptor Proteins, Signal Transducing/metabolism , Atrial Fibrillation/physiopathology , Atrial Fibrillation/genetics , Immunohistochemistry , Nuclear Proteins/genetics , Cell Movement , Connexin 43/genetics , Adaptor Proteins, Signal Transducing/genetics
10.
Acta cir. bras ; 34(10): e201901003, Oct. 2019. tab, graf
Article in English | LILACS | ID: biblio-1054672

ABSTRACT

Abstract Purpose: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury. Methods: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology. Results: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group. Conclusions: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.


Subject(s)
Animals , Reperfusion Injury/metabolism , Connexin 43/deficiency , Disease Models, Animal , Liver/blood supply , Aspartate Aminotransferases/analysis , Reference Values , Time Factors , Reperfusion Injury/pathology , Polymerase Chain Reaction , Mice, Knockout , Connexin 43/analysis , Alanine Transaminase/analysis , Genotyping Techniques , gamma-Glutamyltransferase/analysis , Liver/pathology , Necrosis
11.
Article in Chinese | WPRIM | ID: wpr-773530

ABSTRACT

OBJECTIVE@#To investigate the effect of SRC kinase inhibitor PP2 on the invasion and metastasis of lung cancer A549 cells and explore its molecular mechanism.@*METHODS@#MTT assay was used to evaluate the inhibitory effect of PP2 on the proliferation of A549 cells. Cell scratch and Transwell assays were performed to assess the invasion and metastatic capacity of A549 cells after treatment with 1, 2, 4, 8, and 16 μmol/L PP2 for 24 h. Western blotting was used to detect the expressions of connexin43 (Cx43) and MMP-2 in the cells after small interfering RNA (siRNA)-mediated silencing or overexpression of Cx43; the changes in the cell invasion and metastasis in response to PP2 treatment after Cx43 silencing or overexpression were investigated.@*RESULTS@#MTT assay showed that treatment with PP2 at 2, 4, 8, 16, and 32 μmol/L significantly inhibited the proliferation of A549 cells in a concentration-dependent manner. Treatments with PP2 at 1, 2, 4, 8, and 16 μmol/L for 24 h also concentration-dependently lowered the invasion and metastatic abilities of the cells ( < 0.05). At 4 and 8 μmol/L, PP2 significantly increased the expression level of Cx43 protein and decreased the expression level of MMP-2 protein. Overexpression of Cx43 significantly enhanced the inhibitory effect of PP2 on the cell invasion and metastasis, and Cx43 silencing significantly attenuated the inhibitory effect of PP2 ( < 0.05).@*CONCLUSIONS@#PP2 treatment can suppress the invasion and metastasis of A549 cells possibly by modulating the expression of Cx43.


Subject(s)
A549 Cells , Cell Line, Tumor , Cell Movement , Cell Proliferation , Connexin 43 , Humans , Lung Neoplasms , Neoplasm Invasiveness , Protein Kinase Inhibitors , src-Family Kinases
12.
Article in Chinese | WPRIM | ID: wpr-773488

ABSTRACT

OBJECTIVE@#To investigate the effect of connexin43 (Cx43) protein on autophagy in cisplatin (DDP)-resistant testicular cancer I-10 cells.@*METHODS@#The expression of Cx43 proteins in testicular cancer I-10 cells and I-10/DDP cells were detected with Western blotting. I-10/DDP cells were transfected with a full- length mouse Cx43 vector (mCx43) Lipofectamine, the empty vector or Lipofectamine (blank control group), and the changes in the expressions of LC3 and p62 proteins were determined with Western blotting. mCherry-GFP-LC3B transfection and transmission electron microscopy were used to analyze the changes in autophagy of the cells with Cx43 overexpression.@*RESULTS@#Cx43 was significantly decreased in I-10/DDP cells compared with I-10 cells ( < 0.01). Transfection of the I-10/DDP cells with mCx43 vector resulted in significantly increased Cx43 expression in the cells ( < 0.01) and caused significantly decreased expression of LC3-Ⅱ ( < 0.01) and increased expression of p62 ( < 0.05) as compared with the negative control cells. Both transmission electron microscopy and mCherry-GFP-LC3B transfection showed that the number of autophagosomes was obviously reduced in mCx43-transfected cells as compared with the negative control cells.@*CONCLUSIONS@#Cx43 inhibits autophagy in cisplatin-resistant testicular cancer I-10 /DDP cells.


Subject(s)
Animals , Autophagy , Cell Line, Tumor , Cisplatin , Connexin 43 , Metabolism , Drug Resistance, Neoplasm , Male , Mice , Testicular Neoplasms , Metabolism , Pathology
13.
Article in English | WPRIM | ID: wpr-773419

ABSTRACT

OBJECTIVE@#Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs).@*METHODS@#HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively.@*RESULTS@#Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown.@*CONCLUSION@#Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.


Subject(s)
Caspase 1 , Genetics , Metabolism , Connexin 43 , Genetics , Metabolism , Connexins , Genetics , Metabolism , Gene Expression Regulation , Radiation Effects , Human Umbilical Vein Endothelial Cells , Physiology , Radiation Effects , Humans , Nerve Tissue Proteins , Genetics , Metabolism , Pyroptosis , X-Rays
14.
Article in Chinese | WPRIM | ID: wpr-772077

ABSTRACT

OBJECTIVE@#To investigate the expression of connexin 43 (Cx43) in peripheral blood monocytes (PBMCs) from patients with acute coronary syndrome (ACS) and its clinical implications.@*METHODS@#We prospectively collected the clinical data from 40 patients with ACS including 20 with unstable angina pectoris (UAP) and 20 with acute myocardial infarction (AMI) admitted in our department between January, 2018 and June, 2018, with 20 healthy subjects undergoing routine physical examinations serving as the control group. Peripheral blood samples were obtained from all the participants and plasma and PBMCs were separated. Enzyme-linked immunosorbent assay (ELISA) and turbidimetric inhibition immunoassay (TIIA) were used for analysis of plasma levels of interleukin (IL)-1β and high sensitive C-reactive protein (hs-CRP), respectively; real-time quantitative RT-PCR and Western blotting were used to detect the mRNA and protein levels of Cx43 in the PBMCs.@*RESULTS@#Compared with the control group, the patients with UAP showed significantly increased plasma levels of IL-1β and hs-CRP ( < 0.001) and obviously elevated expressions of Cx43 at both mRNA and protein levels in the PBMCs ( < 0.001). Compared with the patients with UAP, the patients with AMI had significantly higher plasma IL-1β and hs-CRP levels ( < 0.001 and < 0.01) but lower expression levels of Cx43 in the PBMCs ( < 0.05).@*CONCLUSIONS@#Patients with UAP and AMI have activated inflammatory responses and reverse changes in Cx43 expression in the PBMCs, suggesting the different roles of Cx43 in the pathogenic mechanisms of different types of ACS.


Subject(s)
Acute Coronary Syndrome , Angina, Unstable , C-Reactive Protein , Connexin 43 , Humans , Monocytes
15.
Article in English | WPRIM | ID: wpr-761783

ABSTRACT

The present study was designed to examine the effect of heme oxygenase-1 (HO-1) induction by cobalt protoporphyrin (CoPP) on the cardiac functions and morphology, electrocardiogram (ECG) changes, myocardial antioxidants (superoxide dismutase [SOD] and glutathione [GSH]), and expression of heat shock protein (Hsp) 70 and connexin 43 (Cx-43) in myocardial muscles in isoproterenol (ISO) induced myocardial infarction (MI). Thirty two adult male Sprague Dawely rats were divided into 4 groups (each 8 rats): normal control (NC) group, ISO group: received ISO at dose of 150 mg/kg body weight intraperitoneally (i.p.) for 2 successive days; ISO + Trizma group: received (ISO) and Trizma (solvent of CoPP) at dose of 5 mg/kg i.p. injection 2 days before injection of ISO, with ISO at day 0 and at day 2 after ISO injections; and ISO + CoPP group: received ISO and CoPP at a dose of 5 mg/kg dissolved in Trizma i.p. injection as Trizma. We found that, administration of ISO caused significant increase in heart rate, corrected QT interval, ST segment, cardiac enzymes (lactate dehydrogenase, creatine kinase-muscle/brain), cardiac HO-1, Hsp70 with significant attenuation in myocardial GSH, SOD, and Cx-43. On the other hand, administration of CoPP caused significant improvement in ECG parameters, cardiac enzymes, cardiac morphology; antioxidants induced by ISO with significant increase in HO-1, Cx-43, and Hsp70 expression in myocardium. In conclusions, we concluded that induction of HO-1 by CoPP ameliorates ISO-induced myocardial injury, which might be due to up-regulation of Hsp70 and gap junction protein (Cx-43).


Subject(s)
Adult , Animals , Antioxidants , Body Weight , Cobalt , Connexin 43 , Connexins , Creatine , Electrocardiography , Glutathione , Hand , Heart Rate , Heat-Shock Proteins , Heme Oxygenase-1 , Heme , HSP70 Heat-Shock Proteins , Humans , Isoproterenol , Male , Muscles , Myocardial Infarction , Myocardium , Oxidoreductases , Rats , Tromethamine , Up-Regulation
16.
Chinese Medical Journal ; (24): 2354-2361, 2019.
Article in English | WPRIM | ID: wpr-803007

ABSTRACT

Background@#In our previous paper, we demonstrated that Connexin 43 (CX43) was highly expressed in bladder cancer (BC) tissues. But the molecular mechanism about microRNAs (miRNAs) regulation upstream of CX43 in BC has not been well elucidated and remains to be further studied. MicroRNA-139-5p (miR-139-5p) is a tumor suppressor in progression of multifarious cancers including BC. Nevertheless, the underlying mechanisms of CX43/miR-139-5p in tumorigenesis of BC are still not well illustrated. The specific objective of our study was to inquiry the effect of CX43/miR-139-5p on BC progression and its underlying mechanism.@*Methods@#The bioinformatics analysis softwares were applied to predict the miRNAs in the upstream of CX43. First, the expression levels of miR-139-5p in BC tissues (tumor) and paracancer tissues (normal) were investigated using the data from The Cancer Genome Atlas database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of miR-139-5p in three human BC cell lines 5637, T24, ECV-304 and a human bladder epithelial immortalized cell line SV-HUC-1 (normal control). Then si-CX43, si-control, miR-139-5p mimic, and its negative control (NC) were transfected into BC cell line ECV-304. The relationship of miR-139-5p and CX43 was analyzed by dual-luciferase reporter assay. The qRT-PCR and Western blotting were used to test the mRNA and protein expression level of CX43. The proliferation of ECV-304 and T24 cells were examined by cell counting kit-8. The migration and invasion of ECV-304 cells were tested by transwell assay. To determine whether miR-139-5p would affect cell proliferation, migration and invasion by targeting CX43, we executed the rescue assay. The comparison between two groups was analyzed by Student’s t test, and comparisons among multiple samples were performed by oneway analysis of variance and a Bonferroni post hoc test.@*Results@#The expression of miR-139-5p was remarkably down-regulated in BC tissues (tumor vs. normal, 2.286 ± 0.017 vs. 3.211 ± 0.034, t= 11.540, P < 0.0001) and cell lines (P < 0.01 in all BC cell lines). Besides, we also indicated that over-expression of miR-139-5p reduced the proliferation of ECV-304 (P = 0.001) and T24 cells (P = 0.005). Moreover, miR-139-5p over-expression weakened the invasion (P = 0.001) and migration (P = 0.001) of ECV-304 cells. Furthermore, the relative luciferase activity of CX43-wild type construct was distinctly lessened by up-regulation of miR-139-5p (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.916 ± 0.063 vs. 0.356 ± 0.048, t = 7.085, P = 0.002), nevertheless the activity of CX43-mutant type construct was untouched (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.918 ± 0.057 vs. 0.878 ± 0.039, t= 0.577, P = 0.595). Finally, the rescue assay revealed that CX43 deletion enhanced the depressor effect of miR-139-5p on ECV-304 cell proliferation (P < 0.01), invasion (P = 0.028), and migration (P = 0.014).@*Conclusion@#MiR-139-5p, as a tumor-suppressor, repressed cell proliferation, invasion, and migration in BC, which might be achieved by regulating CX43.

17.
Article in Chinese | WPRIM | ID: wpr-801373

ABSTRACT

Objective@#To explore the relationship between gap junction and glucose uptake of astrocytes under oxygen-glucose deprivation(OGD) and reperfusion.@*Methods@#Cerebral cortical astrocyte from 1 day newborn SD rats were undergone the primary culture. The ischemia cell model was established by OGD. This experiment were divided into control group, OGD group and OGD+ CBX group.After different reperfusion time (0 h, 12 h 24 h and 48 h), the glucose uptake of astrocyte was measured by 2-NBDG through flow cytometry analysis and connexin 43(Cx43) gap junction plaques was detected using immunofluorescene.@*Results@#Compared with the control group, the glucose uptake of astrocyte was up-regulated induced by OGD following different reperfusion time.The glucose uptake of OGD group was (2.32±0.43)nmol/μgDNA in 24 hours reperfusion time and was (0.95±0.28)nmol/μgDNA in control group. The up-regulation was up to 2.63-fold increase (t=13.99, P=0.0024) in 24 hours after reperfusion.Compared with the control group, the Cx43 gap junction number was up to 2.5- fold increase(t=11.34, P=0.003) and the size was 1.85-fold increase (t=10.27, P=0.004) in 24 h reperfusion. The glucose uptake of astrocyte after OGD was reduced by CBX and the decrease was 42% in 48 h after reperfusion.@*Conclusion@#Those results urges us consider the clinical treatment for interfering with Cx43 gap junction.

18.
Article in Chinese | WPRIM | ID: wpr-905521

ABSTRACT

Objective:To observe the effects of exercise preconditioning on blood-brain barrier (BBB) permeability, and connexin 43 (Cx43) and pannexin 1 (Panx1) protein after cerebral ischemia-reperfusion injury in rats. Methods:Fifty-four male Sprague-Dawley rats were randomly divided into sham group (n = 18), model group (n = 18) and exercise preconditioning group (n = 18). The exercise preconditioning group was trained with treadmill for three weeks before modeling. The middle cerebral arteries were occluded in the model group and the exercise preconditioning group using the modified Koizumi suture. After reperfusion of 24 hours, the rats were assessed with modified Neurological Severity Score (mNSS). The permeability of BBB was observed with Evans blue (EB). The expression of Cx43 and Panx1 was detected with Western blotting and immunohistochemistry in the ischemic tissues. Results:Compared with the model group, the mNSS score decreased in the exercise preconditioning group (P < 0.05), while the Evans blue content and the expression of Cx43 and Panx1 decreased (P < 0.05), as well as the the positive areas of Cx43 and Panx1 (P < 0.05). Conclusion:Exercise preconditioning can improve the permeability of BBB in cerebral ischemia-reperfusion rats, which may associate with down-regulation of Cx43 and Panx1, to protect brain from injury.

19.
Chinese Pharmacological Bulletin ; (12): 1528-1534, 2019.
Article in Chinese | WPRIM | ID: wpr-857097

ABSTRACT

was observed by patch clamp. Results Cx32 or Cx26 expression and GJ function were induced by doxycycline (Dox, the promotor for PBI plasmid) in transfected Hela cells. MiR-124 reduced the proliferation of Hela cells, dox incubation alone did not affect Hela cell growth, and also had no effect on anti-tumor effect of miR-124 when combined with miR-124 transfection. Compare with U 87shRNA-Cx43 , the Cx43 expression and GJ function significantly decreased in U87shRNA-Cx43. Similar to the effect on Hela cells, MiR-124 also reduced U87 cell growth. Reducing Cx43 expression did not influence U87 cell proliferation, but attenuated the growth-inhibition effect of miR-124 when combined with miR-124 transfection. Under the microscope, the transfer of fluorescence-labelled miR-124 from "donor" cell to adjacent " non-injection " cell was observed. Conclusions The role of GJ on anti-tumor effect of miR-124 possesses connexin heterogeneity. Compare with Cx26 or Cx32, GJ composed of Cx43 has more obvious effect, which may be related to the maximum permeability of junction channel to miR-124.

20.
Article in Chinese | WPRIM | ID: wpr-824249

ABSTRACT

Objective To explore the relationship between gap junction and glucose uptake of astrocytes under oxygen-glucose deprivation(OGD) and reperfusion.Methods Cerebral cortical astrocyte from 1 day newborn SD rats were undergone the primary culture.The ischemia cell model was established by OGD.This experiment were divided into control group,OGD group and OGD + CBX group.After different reperfusion time (0 h,12 h 24 h and 48 h),the glucose uptake of astrocyte was measured by 2-NBDG through flow cytometry analysis and connexin 43(Cx43) gap junction plaques was detected using immunofluorescene.Results Compared with the control group,the glucose uptake of astrocyte was up-regulated induced by OGD following different reperfusion time.The glucose uptake of OGD group was (2.32 ± 0.43)nmol/μgDNA in 24 hours reperfusion time and was (0.95±0.28)nmol/μgDNA in control group.The upregtulation was up to 2.63-fold increase (t=13.99,P=0.0024) in 24 hours after reperfusion.Compared with the control group,the Cx43 gap junction number was up to 2.5-fold increase(t=11.34,P=0.003) and the size was 1.85-fold increase (t=10.27,P=0.004) in 24 h reperfusion.The glucose uptake of astrocyte after OGD was reduced by CBX and the decrease was 42% in 48 h after reperfusion.Conclusion Those results urges us consider the clinical treatment for interfering with Cx43 gap junction.

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