Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.
Animals ; Rats ; Myocytes, Cardiac ; Kruppel-Like Factor 6 ; Connexin 43 ; Desmin ; Cell Differentiation ; Azacitidine/pharmacology* ; Mesenchymal Stem Cells ; RNA, Messenger ; MicroRNAs

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.
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*

The present study was designed to examine whether Ramipril (an inhibitor of angiotensin-converting enzyme) affected spontaneous hypertension-induced injury of cerebral artery by regulating connexin 43 (Cx43) expression. Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were randomly divided into WKY, WKY + Ramipril, SHR, and SHR + Ramipril groups (n = 8). The arterial pressure was monitored by the tail-cuff method, and vascular function in basilar arteries was examined by pressure myography. Hematoxylin-eosin (HE) staining was used to show vascular remodeling. The expression and distribution of Cx43 was determined by using immunofluorescence and immunohistochemistry analysis. The protein and mRNA levels of Cx43 were examined by Western blot and real-time PCR analysis, respectively. The results showed that chronic Ramipril treatment significantly attenuated blood pressure elevation (P < 0.01, n = 8) and blood vessel wall thickness in SHR (P < 0.01, n = 8). The cerebral artery contraction rate in the SHR group was higher than that in the WKY group (P < 0.05, n = 8). The cerebral artery contraction rate in the SHR + Ramipril group was lower than that in the SHR group (P < 0.05, n = 8). Pretreatment with 2-APB (Cx43 non-specific blocker) or Gap26 (Cx43 specific blocker) significantly decreased the vasoconstriction rate, while pretreatment with AAP10 (Cx43 non-specific agonist) significantly increased the vasoconstriction in the SHR + Ramipril group (P < 0.05, n = 8). In addition, the expression of Cx43 mRNA and protein in cerebral arteries of SHR group was higher than that of WKY group (P < 0.05, n = 8). The mRNA and protein expression of Cx43 in cerebral arteries of SHR + Ramipril group was significantly lower than that of SHR group (P < 0.05, n = 8). These results suggest that Ramipril can down-regulate the expression of Cx43 mRNA and protein in cerebral arterial cells of SHR, lower blood pressure, promote vasodilation, and improve arterial damage and vascular dysfunction caused by hypertension.
Animals ; Blood Pressure ; Cerebral Arteries ; drug effects ; metabolism ; Connexin 43 ; metabolism ; Hypertension ; drug therapy ; Ramipril ; pharmacology ; Random Allocation ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Vascular Remodeling

OBJECTIVE: To investigate the effects of total flavonids of astragalus(TFA) on arrhythmia, endoplasmic reticulum stress and connexcin in mice with viral myocarditis and to clarify the mechanisms of TFA against viral myocarditis complicated with arrhythmia. METHODS: Thirty-six male Balb/c mice were randomly divided into control group, viral myocarditis group and total flavonoids group (=12). The mice of viral myocarditis were intraperitonealy injected with 0.1 ml/day 10-950 TCID CVB3 for 3 days. The mice of TFA group were intraperitoneal injected with 0.1 ml/day 10-950 TCID CVB3 for 3 days and treated with 0.1ml, 20 mg/L TFA by tail vein injection. At the end of the experiment, arrhythmia was detected by electrocardiogram, the heart of mice were stained by HE, the expressions of glucose-regulated protein 78(GRP78), endoplasmic reticulum stress signaling pathway factor activating transcription factor 4(ATF4) and connexcin 43(Cx43) were detected by Western blot. RESULTS: The expressions of GRP78 and ATF4 were increased and the expression of Cx43 was decreased in viral myocarditis, while TFA inhibited these effect of viral myocarditis in heart of mice. CONCLUSIONS The antiarrhythmic effect of TFA may be related to the alleviation of endoplasmic reticulum stress and the increase of Cx43 expression.
Activating Transcription Factor 4 ; metabolism ; Animals ; Arrhythmias, Cardiac ; drug therapy ; Astragalus Plant ; chemistry ; Connexin 43 ; metabolism ; Coxsackievirus Infections ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Flavonoids ; pharmacology ; Heat-Shock Proteins ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; Myocardium

Increasing evidence suggests that spinal microglia regulate pathological pain in males. In this study, we investigated the effects of several microglial and astroglial modulators on inflammatory and neuropathic pain following intrathecal injection in male and female mice. These modulators were the microglial inhibitors minocycline and ZVEID (a caspase-6 inhibitor) and the astroglial inhibitors L-α-aminoadipate (L-AA, an astroglial toxin) and carbenoxolone (a connexin 43 inhibitor), as well as U0126 (an ERK kinase inhibitor) and D-JNKI-1 (a c-Jun N-terminal kinase inhibitor). We found that spinal administration of minocycline or ZVEID, or Caspase6 deletion, reduced formalin-induced inflammatory and nerve injury-induced neuropathic pain primarily in male mice. In contrast, intrathecal L-AA reduced neuropathic pain but not inflammatory pain in both sexes. Intrathecal U0126 and D-JNKI-1 reduced neuropathic pain in both sexes. Nerve injury caused spinal upregulation of the astroglial markers GFAP and Connexin 43 in both sexes. Collectively, our data confirmed male-dominant microglial signaling but also revealed sex-independent astroglial signaling in the spinal cord in inflammatory and neuropathic pain.
2-Aminoadipic Acid ; toxicity ; Animals ; Anti-Inflammatory Agents ; therapeutic use ; Astrocytes ; pathology ; Carbenoxolone ; pharmacology ; Caspase 6 ; deficiency ; metabolism ; Connexin 43 ; metabolism ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Male ; Mice ; Mice, Transgenic ; Microglia ; pathology ; Minocycline ; therapeutic use ; Neuralgia ; chemically induced ; drug therapy ; pathology ; Pain Measurement ; Phenylurea Compounds ; pharmacology ; Sex Characteristics ; Spinal Cord ; pathology ; Time Factors

OBJECTIVETo investigate the effects of the quinoline derivative PQ1 combined with cisplatin on the proliferation and gap junction communication of prostate cancer PC3 cells.

METHODSWe cultured in vitro prostate cancer PC3 cells and divided them into DMSO blank control, cisplatin control, and cisplatin (10 mg/ml) plus PQ1 (1, 2, 5, 10, and 15 μmol/L) groups. We measured the proliferation of the prostate cancer PC3 cells, determined the expressions of the connexin 43 (Cx43) mRNA and protein by RT-PCR and Western blot, and compared the indexes among different groups.

RESULTSCisplatin combined with PQl at 1 - 10 μmol/L significantly inhibited the proliferation of the PC3 cells and the inhibition rate rose in a concentration- and time-dependent manner, from (48.72 ± 0.98)% vs (50.33 ± 0.62)% at 0 μmol/L to (77.38 ± 1.12)% vs (83.50 ± 1.05)% at 15 μmol/L at 24 and 48 hours (P < 0.05). Compared with the cisplatin control, cisplatin combined with PQ1 at 1, 2, 5, 10, and 15 μmol/L increased the expression of Cx43 mRNA from 0.379 ± 0.113 to 0.669 ± 0.031, 0.831 ± 0. 127, 0.769 ± 0.100, 0.532 ± 0.086, and 0.475 ± 0.134, respectively (P < 0.05), and cisplatin combined with PQ1 at 1, 2, 5, and 10 μmol/L elevated that of Cx43 protein from 0.138 ± 0.146 to 0.263 ± 0.111, 0.306 ± 0.152, 0.415 ± 0.280, and 0.643 ± 0.310, respectively (P < 0.05).

CONCLUSIONThe quinoline derivative PQ1 can promote the gap junction communication of prostate cancer PC3 cells and enhance the killing effect of cisplatin on PC3 cells by upregulating the expressions of Cx43 mRNA and protein.

Aminoquinolines ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Connexin 43 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Gap Junctions ; drug effects ; physiology ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; physiopathology ; RNA, Messenger ; metabolism ; Time Factors

ObjectiveTo investigate the effect of salidroside on the expression of the connexin43 (Cx43) protein in the corpus cavernosum smooth muscle cells (CCSMCs) of hypoxic SD rats.

METHODSCCSMCs were cultured in vitro and identified by immunofluorescence staining. The cells were divided into six groups: normal control (21% O2), hypoxia (1% O2), hypoxia+salidroside (HS) 8 μg/ml,HS 16 μg/ml, HS 32 μg/ml, and HS 64 μg/ml, and cultured for 48 hours. Then the relative expression of Cx43 in different groups was detected by Western blot.

RESULTSThe in vitro cultured CCSMCs grew well and 90% of the cells showed positivity for α-SMA and desmin on immunohistochemistry. Salidroside ≤64 μg/ml produced no obvious toxicity on the CCSMCs. The expressions of Cx43 and phosphorylated proteins were dramatically increased in the hypoxia group as compared with the normal control (P<0.01 and P<0.05). The HS groups all showed significantly higher expression of Cx43 than the hypoxia group (P<0.01), but the phosphorylation rate of the Cx43 proteins was remarkably decreased (P<0.01).

CONCLUSIONSHypoxia increases the expression of Cx43 in the CCSMCs of SD rats. Salidroside ≤64 μg/ml cannot reverse the hypoxia-induced change but can reduce the dephosphorylation of Cx43 in CCSMCs. It is deduced that salidroside can protect CCSMCs by decreasing the phosphorylation of Cx43 and suppressing hypoxia-induced formation of the gap junction channel.

Actins ; metabolism ; Animals ; Cell Hypoxia ; Cells, Cultured ; Connexin 43 ; metabolism ; Glucosides ; pharmacology ; Male ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Penis ; drug effects ; metabolism ; Phenols ; pharmacology ; Phosphorylation ; drug effects ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of total flavonoids of astragalus on the expression of endoplasmic reticulum chaperone, calumenin and connecxin 43 (CX43) in suckling mouse myocardium with myocarditis caused by coxsackievirus B3 (CVB3).

METHODSThe primary culture of suckling mouse myocardium cells were randomly divided into control group, CVB3 infected group and total flavonoids of astragalus group. Firstly, to confirm the identity of the suckling mouse myocardium, α-SMA was monitored by immunohistochemistry method. Then the protein expression changes of endoplasmic reticulum chaperone-glucose regulatory protein 78 ( GRP78), calumenin and CX43 were detected by Western blot.

RESULTS(1) Compared with that of the control group, the GRP78 expression level in CVB3 infected group was improved, the expression levels of calumenin and CX43 were all reduced. (2) Compared with that of CVB3 infected group, GRP78 expression level was decreased, and the expression levels of calumenin and CX43 were increased in total flavonoids of astragalus group.

CONCLUSIONCVB3 infection may cause endoplasmic reticulum stress of rat myocardium cells by increasing the expression of GRP78 and decreasing the expression of calumenin and CX43. On the other hand, total flavonoids of astragalus can reduce the expression of GRP78 and increase the expression of calumenin and CX43.The results of this experiment may be closely related to the effects of anti-arrhythmia with viral myocarditis caused by CVB3.

Animals ; Astragalus Plant ; chemistry ; Blotting, Western ; Calcium-Binding Proteins ; metabolism ; Cells, Cultured ; Connexin 43 ; metabolism ; Coxsackievirus Infections ; drug therapy ; Endoplasmic Reticulum ; metabolism ; Endoplasmic Reticulum Stress ; drug effects ; Flavonoids ; pharmacology ; Heat-Shock Proteins ; metabolism ; Mice ; Myocarditis ; drug therapy ; virology ; Myocardium ; cytology ; Myocytes, Cardiac ; drug effects ; virology ; Rats

OBJECTIVETo investigate the effects of simvastatin on the proliferation and apoptosis of prostatic epithelial RWPE-1 cells.

METHODSRWPE-1 cells cultured in vitro were treated with simvastatin at 0, 10, 20, and 40 μmol/L for 24, 48, and 72 hours followed by determination of their proliferation by MTT assay, and their apoptosis by flow cytometry. The mRNA and protein expressions of Bcl-2, Bax, and Cx43 were detected by fluorescence quantitative RT-PCR and Western blot, respectively.

RESULTSAfter 72 hours of treatment with simvastatin at 10, 20, and 40 μmol/L, the inhibition rates of the RWPE-1 cells were (21.07 ± 6.41)%, (34.87 ± 9.65)%, and (47.18 ± 10.88)%, respectively, significantly higher than (1.21 ± 0.54)% in the control group (P < 0.05) and in a dose-dependent manner (P < 0.05); the cell apoptosis rates were (0.066 ± 0.016)%, (0.126 ± 0.023)%, and (0.192 ± 0.025)%, respectively, remarkably higher than (0.015 ± 0.005)% in the control (P < 0.05) and also in a dose-dependent manner (P < 0.05); the mRNA and protein expressions of Bcl-2 were decreasing while those of Bax and Cx43 increasing with the increased concentration of simvastatin (P < 0.05). The expression of Cx43 was correlated negatively with that of Bcl-2 but positively with that of Bax.

CONCLUSIONSimvastatin inhibits the proliferation of prostate epithelial cells and induce their apoptosis by acting on the gap junctional intercellular communication.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Connexin 43 ; metabolism ; Drug Administration Schedule ; Epithelial Cells ; drug effects ; physiology ; Humans ; Hypolipidemic Agents ; pharmacology ; Male ; Prostate ; cytology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; metabolism ; Simvastatin ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the protective effect of propofol against focal cerebral ischemia/reperfusion (I/R) injury in rats and its relation with gap junction.

METHODSSeventy adult male SD rats were randomly divided into sham-operated group, I/R group, low-, moderate-, and high-dose propofol groups (25, 50, 100 mg/kg; P25, P50, P100 groups, respectively), I/R+CBX group and P100+CBX group. Thread occlusion was used to induce middle cerebral artery occlusion (MCAO) in the mice for 2 h followed by reperfusion for 24 h. Longa's scores were used to evaluate the neurological behavior of the rats. TTC staining was used to measure the cerebral infarction volume and Western blotting was performed to detect the expressions of Cx43, PKC, Bax, and Bcl-2 in the brain of the rats.

RESULTSCompared with the I/R group, the rats pretreated with moderate and high doses of propofol showed significantly reduced neurological behavior scores and cerebral infarction volume percentage, and the effect was more obvious in high-dose propofol pretreatment group. CBX obviously enhanced the protective effect of propofol against I/R injury. Compared with those in the sham-operated group, the protein expression of Cx43 and the Bax/Bcl-2 ratio were increased and the protein expression of PKC was reduced in I/R group, and these changes were significantly reversed by high-dose propofol pretreatment; the effects of propofol were further enhanced by CBX.

CONCLUSIONThe protective effect of propofol against cerebral I/R injury may involve the inhibition of the gap junction via PKC signaling and by reducing the Bax/Bcl-2 ratio.

Animals ; Brain ; metabolism ; Brain Ischemia ; prevention & control ; Connexin 43 ; metabolism ; Gap Junctions ; Infarction, Middle Cerebral Artery ; Male ; Propofol ; pharmacology ; Protein Kinase C ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; Signal Transduction ; bcl-2-Associated X Protein ; metabolism