A comparative computational approach on the most deleterious missense variant in Connexin 43 protein and its potent inhibitor analysis

Intercellular communication between the cell plays an essential role in cell growth and cell formation, including migration, metabolism, and cell differentiation. Cell function and tissue homeostasis are maintained through gap junction intercellular communication (GJIC), thus regulating connexin hemichannels. Mis regulation of such connexin, especially connexin (Cx) 43, affects a comprehensive process, including cell differentiation, inflammation, and cell death. Mis regulation may be due to the missense variant in Cx43. Thus, we screened the complete set of mutations from public mutational databases and obtained 219 missense variants, which were then classified based on their pathogenicity, functional impact, stability, conservation, and physiochemical properties. Variant L214P was scrutinized to have the most deleterious, which was then modelled using the I-TASSER server and performed molecular docking analysis to screen potent inhibitors. The compound Kanamycin, Ginsenoside, and Astragaloside IV have better interactions with Cx43 mutant with a maximum of 5 hydrogen bonds. Ginsenoside is a compound that follows a Lipinski rule of five. Thus, the result obtained from this study suggests that Ginsenoside would be a better potent inhibitor for native and mutant Cx43.

Effect of Modified Shuyuwan on Hippocampal Myelin Sheath Injury in Vascular Dementia Rats Through Cx43/Glu/AMPAR Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of modified Shuyuwan （SYW） on hippocampal myelin sheath injury in vascular dementia （VD） model rats. MethodSixty male SD rats of SPF grade were randomly divided into sham operation group， model group， and high-， medium- and low-dose modified SYW groups， with 12 rats in each group. The VD model was induced by bilateral carotid artery ligation in rats of the groups except for those of the sham operation group. After modeling， rats were screened by the water maze test， followed by drug treatment by gavage. Specifically， rats in the modified SYW groups were treated with modified SYW at 10， 5， 2.5 g·kg-1·d-1， accordingly， and those in other groups were administered with the same amount of normal saline. After intragastric administration for 28 days， the spatial learning and memory abilities of rats were detected by the water maze test. The hippocampal neuron structure was observed by hematoxylin-eosin （HE） staining. The content of hippocampal tumor necrosis factor （TNF）-α， interleukin-6 （IL-6）， and glutamate （Glu） was observed by biochemical detection. The hippocampal expression of myelin basic protein （MBP）， astrocyte activation marker glial fibrillary acidic protein （GFAP）， and connexin 43 （Cx43） was detected by immunofluorescence detection. The myelin sheath structure in the hippocampus was observed by the electron microscope. The α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor （AMPAR） and Cx43 protein expression was detected by Western blot. ResultCompared with the sham operation group， the model group showed prolonged escape latency （P<0.01）， decreased times of crossing the original platform and percentage of target quadrant detention time （P<0.01）， disordered neuron structure in the hippocampal CA1 region， loose myelin sheath lamella with blurry edge， up-regulated expression levels of TNF-α， IL-6， and Glu in the hippocampal CA1 region， especially Glu （P<0.01）， reduced expression of AMPAR （P<0.01）， increased protein expression of p-AMPAR and Cx43 （P<0.01）， significantly dwindled protein expression of MBP in the myelin sheath， and enhanced fluorescence co-labeled by GFAP and Cx43. Compared with the model group， the modified SYW groups showed shortened escape latency （P<0.05）， increased times of crossing the original platform and percentage of target quadrant detention time （P<0.05）， closely arranged hippocampal neuron structure， denser myelin sheath lamella with clear edge， down-regulated expression levels of TNF-α， IL-6， and Glu in the hippocampal CA1 region， especially Glu （P<0.01）， up-regulated AMPAR （P<0.01）， reduced protein expression of p-AMPAR and Cx43， especially in the high-dose group （P<0.01）， significantly elevated protein expression of MBP in the myelin sheath， and weakened fluorescence co-labeled by GFAP and Cx43， especially in the high-dose group. ConclusionModified SYW can improve the learning and memory abilities of VD rats， and the mechanism may be related to the inhibition of Cx43 expression， reduction of the release of Glu， inhibition of AMPAR-mediated inflammatory response to reduce the production of astrocyte marker GFAP， and promotion of the expression of MBP protein to alleviate myelin injury.

Changes of connexin 43 in irradiated human vascular endothelial cells and its influence on cell stiffness / 中华放射医学与防护杂志

Objective:To investigate the changes of connexin 43 (Cx43) in human umbilical vein endothelial cells (HUVEC) after X-ray irradiation and its influence on the stiffness of irradiated cells.Methods:Western blot was used to detect the expression of Cx43 in HUVEC cells at different time points (0, 6, 12, 24 and 48 h) after different doses of X-ray irradiation (0, 2.5, 5, 10 and 20 Gy), and the phosphorylation levels of three phosphorylation sites (Ser279/282, Ser368 and Tyr265) of Cx43 at different time points (3, 6, 24 and 48 h) after 0, 5 and 10 Gy irradiation. The distribution of Cx43 protein in the irradiated HUVEC cells was detected by immunofluorescence. The stiffness changes of cells were detected by atomic force microscopy (AFM) at the depths of 50, 100 and 200 nm.Results:The expression of Cx43 in HUVEC cells was reduced at 6, 12, 24 and 48 h after 10 Gy X-ray irradiation( t=3.262, 3.708, 3.686, 6.825, P<0.05)and this decrease had a dose dependent manner at 24 h after 2.5, 5, 10 and 20 Gy irradiation ( t=3.034, 10.720, 13.130, 13.650, P<0.05). At 24 h after 5, 10 and 20 Gy X-ray irradiation, the distribution of Cx43 in HUVEC cells was transported from intercellular gap junctions to nucleus and perinuclear region. At 24-48 h after irradiation, the phosphorylation level of Ser368 at Cx43 increased and in a dose dependent manner. At 24 h after irradiation, the stiffness of the irradiated cells decreased significantly under the conditions of 100 and 200 nm ( t=3.362, 5.122, P<0.05), and recovered with overexpression of Cx43 ( t=2.674, 4.398, P<0.05). Conclusions:X-ray irradiation leads to the phosphorylation of Ser368 at Cx43, which promotes the degradation and nucleus/perinuclear translocation of Cx43 and reduces the stiffness of HUVEC. Increasing the expression level of Cx43 is helpful to the stiffness recovery of irradiated vascular endothelial cells, suggesting that Cx43 may be a potential target for regulating radiation injury of vascular endothelial cells.

Effect of Buyang Huanwu Tang on Synaptic Structural Plasticity After Cerebral Ischemia-reperfusion in Rats / 中国实验方剂学杂志

<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^-1), Gap26 was intraperitoneally injected once a day since the third day after surgery (25 g·kg^-1). Brain was taken out at the 7^th 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.

Formation of gap junctions between adipose stem cells-derived Schwann cells in a rat model of dyskinesia induced by brain injury / 南方医科大学学报

OBJECTIVE@#To investigate the formation of gap junctions between Schwann cells derived from differentiated adipose stem cells implanted in a rat model of dyskinesia induced by brain injury and its positive effect in promoting functional recovery of the rats.@*METHODS@#In a rat model of hemiplegia induced by motor cortex injury, adipose stem cells or Schwann cells differentiated from adipose stem cells, either with or without RNAi-mediated silencing of Cx43, were transplanted orthotopically in the lesion. The recovery of the motor function of the rats was observed and scored after the transplantation. Rat brain tissues were sampled to detect the expressions of nerve growth factor (NGF) using Western blotting and RT-PCR.@*RESULTS@#All the 3 cell transplantation therapies obviously improved the motor function scores of the rats as compared with the control rats. The expression of NGF in the brain tissue was significantly lower in the control group than in the cell transplantation groups. NGF expression in the brain tissues of rats receiving transplantation of Schwann cells with Cx43 gene silencing was lower than that in rats receiving Schwann cells without Cx43 silencing, and was similar with that in rats transplanted with adipose stem cells. The results of RT-PCR showed that NGF mRNA level in the control group was significantly lower than that in the other 3 groups. NGF mRNA expression was the highest in Schwann cell group without Cx43 silencing, followed by adipose stem cell group, and then by Schwann cell group with Cx43 silencing.@*CONCLUSIONS@#In the rat model of dyskinesia induced by brain injury, transplantations of adipose stem cells and adipose stem cells-derived Schwann cells both promote the functional recovery of brain damage, in which gap junction protein Cx43 plays an important role to promote functional gap junction formation possibly by enhancing NGF expression.

Role of gap junction protein connexin43 in pathogenesis of neuro-disorders / 中国药理学通报

Gap junction (GJ), which mainly consists of connexins, is a linking method between neighboring cells. Neighboring cells exchange substances, energy and information by gap junction intercellular communication mediated by GJ. Particularly, Cx43 protein is one of the most important members of Cx family. The study in recent years demonstrated the features of Cx43 protein and its important roles in the pathogenesis of neurological disorders, tumors, cardiovascular risks. This article summarizes the structure, functions, coding gene, structure analysis, synthesis, membrane localization, regulation of Cx43. Furthermore, this article explains the physiological functions of Cx43 in human bodies and its important roles in the pathogenesis of related neurological disorders.

Genistein Improves the Major Depression through Suppressing the Expression of miR-221/222 by Targeting Connexin 43

OBJECTIVE: Recent studies have indicated the possibility that genistein may improve depression via regulating the expression of miR-221/222. This study is to explore whether genistein could improve depression by altering miR-221/222 levels and investigate the possible mechanisms involved in the improvement effect of genistein. METHODS: The animal model of depression was established through unpredictable chronic mild stress. Nest building test and splash test were adapted to evaluate the effects of genistein on depressive symptoms in mice. qRT-PCR and western blot analysis were used to detect the expression of miR-221/222 and connexin 43 (Cx43) in the prefrontal cortex of the mice. In vitro, U87-MG astrocytes were treated with genistein and the expression of miR-221/222 and Cx43 was measured. The dual-luciferase reporter assay was used to verify whether Cx43 was a direct target of miR-221/222. RESULTS: The behavioral tests showed that genistein could significantly reduce depression symptoms of mice, and this remission was not affected by gender. Genistein in vivo and in vitro could reduce increased levels of miR-221 and miR-222 in the prefrontal cortex of depressed mice, while upregulate Cx43 expression. Dual-luciferase reporter assay suggested Cx43 was directly regulated by miR-221/222 in astrocytes. CONCLUSION: Genistein can play its antidepressant effect through down-regulating miR-221/222 by targeting Cx43.

Effects of Shuwei Decoction on the expressions of Beclin-1, mTOR, Cx43 Protein and mRNA in gastrointestinal smooth muscle of rats with functional dyspepsia due to liver depression and spleen deficiency / 中成药

AIM To investigate the therapeutic action and mechanism of Shuwei Decoction (Bupleuri Radix,Cyperi Rhizoma,Atractylodis macrocephalae Rhizoma,etc.) on functional dyspepsia (FD) from the perspectives of autophagy and intercellular gap junction pathway through its impact on the expressions of Beclin-1,mTOR,Cx43 protein and mRNA in gastric antrum pyloric smooth muscle cells of FD model rats.METHODS Sixty Wistar rats were randomly divided into control (blank) group,model group,mosapride group (1.37 mg/kg),low,medium and high dose Shuwei Decoction groups (7.67 g/kg,15.34 g/kg,30.68 g/kg,respectively).FD model rats were prepared according to compound etiological modeling method for 21 days.On the first day after modeling,the rats in each group were given the corresponding medicine for intragastric administration once a day for 14 days.Two hours after the last administration,we procured gastric antrum and pyloric tissues from the sacrificed rats.The expressions of Beclin-1,mTOR and Cx43 protein were detected by Western blot,and so were the expressions of Beclin-1,mTOR and Cx43 mRNA by quantitative real-time PCR (RT-qPCR).RESULTS We observed decreased expression of Beclin-1 protein in low,medium and high dose Shuwei Decoction groups (P ＜ 0.01),and an increased expression of Cx43 protein in high dose Shuwei Decoction group (P ＜ 0.05).In medium and high dose Shuwei Decoction groups,decreased expression of Beclin-1 mRNA (P ＜ 0.01),and increased expression of mTOR and Cx43 mRNA (P ＜ 0.01) were detected as well.CONCLUSION Shuwei Decoction can improve FD by suppressing cell autophagy and enhancing the function of gap junction through up-regulating the expression of mTOR mRNA and Cx43 protein and mRNA,and down-regulating the expression of Beclin-1 protein and mRNA.

Effects of hyperoside on the myocardical activities of ATPases and expressions of Cx43 and Kir2.1 in arrhythmia rats induced by ischemia-reperfusion / 中成药

AIM To investigate the effects of hyperoside,an anti-arrhythmic agent capable of reducing myocardial infarct size,on arrhythmic rats induced by ischemia-reperfusion (I/R) and the corresponding mechanism.METHODS Male SD rats were randomly assigned to sham operation group,model group and hyperoside group (50 mg/kg,n =15).The I/R model was reconstructed by the ligation of left anterior descending coronary artery for 30 min ischemia.Rats in the hyperoside group were injected with 50 mg/kg hyperoside intraperitoneally 10 min before ischemia.Heart rate,mean arterial pressure (MAP) and heart rate systolic blood pressure product (RPP) at time points of 10 min before ischemia (T0),30 min after ischemia (T1),30 min (T2),60 min (T3),120 min (T4) after reperfusion were recorded.ELISA method was used to determine serum CK-MB and cTnI,spectrophotometry to measure Na+-K+-ATPase and Ca2+-Mg2+-ATPase levels,HE staining to observe myocardial tissue changes,immunohistochemistry to investigate Cx43 protein,and Western blot to detect Kir2.1 protein expression.RESULTS At T1,T2,T3 and T4,the model group demonstrated significantly lower HR,MAP and RPP than those in the sham operation group (P ＜ 0.05),whereas the hyperoside group had higher HR,MAP and RPP than the model group.Both hyperoside group and the model group shared significantly higher arrhythmia score,levels of CK-MB and cTnI than the sham operation group (P ＜ 0.05) while their lower activities of Na +-K +-ATPase and Ca2+-Mg2+-ATPase,protein expressions of Cx43 and Kir2.1 than the sham operation group were noticed as well.But the hyperoside group displayed its lower arrhythmia score,levels of CK-MB and cTnI,and yet higher activities of Na +-K +-ATPase and Ca2 +-Mg2 +-ATPase,protein expressions of Cx43 and Kir2.1 than the model group (P ＜0.05).CONCLUSION Hyperoside in improving ventricular arrhythmia of I/R rats may contribute to the activity restoration of Na +-K +-ATPase and Ca2 +-Mg2 +-ATPase,and the up-regulation of Cx43 and Kir2.1 protein expressions.

Role of connexin 43 in X-ray induced apoptosis in human umbilical vein endothelial cells and its mechanism / 中华放射医学与防护杂志

Objective To study the role of Cx43 in X-ray induced apoptosis of HUVEC cells and its mechanism.Methods Flow cytometry was used to detect the apoptosis of HUVEC cells at 48-96 h after 10 Gy X-ray irradiation and at 72 h after irradiation of different doses.Western blot was used to detect the protein expressions of Cx43 and cleaved caspase-3 in HUVEC cells at 72 h after 0,5,10 and 20 Gy irradiation.Small interfering RNA was transfected into HUVEC cells to silence Cx43 expression,the Cx43 bearing plasmid was transfected into cells to overexpress Cx43.The effect of Cx43 knockdown or overexpression on apoptosis induction and cleaved caspase-3 protein expression were detected by flow cytometry and Western blot,respectively.Results The apoptosis of HUVEC increased significantly from 48 h to 96 h after X-ray irradiation and in a dose-dependent manner at 72 h after irradiation.The expression of Cx43 protein was negatively correlated with the dose but the expression of cleaved caspase-3 was positively correlated with the dose in the range of 0-20 Gy.After Cx43 silencing,the proportion of early apoptosis and apoptosis combined with dead cells were significantly higher than that of the siRNA control group(t =3.674,6.375,P ＜ 0.05).After Cx43 overexpression,the proportion of early apoptosis and apoptosis combined with dead cells were significantly lower than that of vector control group(t =9.399,11.190,P ＜ 0.05).The expression of cleaved caspase-3 in the Cx43 silencing group was higher than that in the siRNA control group,but this protein in the Cx43 overexpressed group was lower than that in the vector control group.Conclusions Cx43 may protect X-ray irradiated HUVEC cells from apoptosis by down-regulating the activation of caspase-3.

Farrerol inhibits AngII-induced VSMCs proliferation by reducing Cx43 expression / 中国药理学通报

Aim To study the role of Cx43 in inhibi-tion of AngII-induced vascular smooth muscle cells(VSMCs) proliferation by farrerol. Methods The primary VSMCs were isolated and cultured by direct adherent culture methods. VSMCs were identified by immunohistochemstry. The cells were divided into the following groups:control group,AngII group,AngII+Farrerol group. The cell viability was measured by CCK-8 cell vitality test. The proliferation of VSMCs was measured by the methods of Edu. The cell cycle of VSMCs was detected by flow cytometry. The mRNA levels of Cx43 were measured by Real-time PCR. The protein levels of Cx43 were measured by Western blot. Results 60 μmol·L-1farrerol could significantly de-crease the cell viability and EdU rate of VSMCs in-duced by AngII(P<0.05),which could also prevent the transformation of VSMCs from G0/G1phase to S phase. The results of real-time PCR and Western blot showed that,compared with the model group,Farrerol could significantly reduce the mRNA and protein ex-pression level of Cx43(P <0.01). After the interfer-ence of Cx43 by siRNA, the inhibition of proliferation by farrerol decreased significantly. Conclusion Far-rerol inhibits AngII-induced VSMCs proliferation signif-icantly, which might be associated with reducing the expression of Cx43.

Progress in research on connexin 43 in oral squamous cell carcinoma / 口腔疾病防治

@#Cell gap junctions are a universal form of cellular connection in animal tissue that mediate the exchange of information, energy and material between adjacent cells. Clinical studies have demonstrated that the abnormal expression of the connexin 43 （Cx43） gene is closely associated with carcinogenesis and tumor progression. The present study reviewed relevant studies concerning the association between abnormal expression of Cx43 and oral squamous cell carcinoma as well as communication abnormalities of cell gap junctions.

Effect of celecoxib on the expression of connexin43 in retina of diabetic rats / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the expression of connexin43(Cx43)in retina of Sprague Dawley(SD)rats with diabetes mellitus(DM), and the effects of celecoxib, inhibitor of cyclooxygenase 2(COX-2), on expression of Cx43 in retina of them. <p>METHODS: DM was induced in SD rats by the administration of streptozotocin(STZ). DM rats were divided into two groups: COX-2 treated group(CTG, <i>n</i>=15), and diabetic group(DG, <i>n</i>=15). Normal control group(NCG)consisted of 15 rats was given. Three months' treatment was given after DM development. Sample of retina were made at the end of 3mo. Expression of Cx43 in retina were investigated by immunohistochemistry. Expression of Cx43 mRNA in retina were analyzed by RT-PCR. <p>RESULTS: Cx43 was expressed in the ganglion cell layer, the nerve fiber layer, the inner plexiform layer, and the pigment epithelium layer of retina with a different degree. Computer image analysis showed that celecoxib had elevated the expression of Cx43 in experimental diabetic rats, gray values were: 0.233±0.025, 0.124±0.014, 0.197±0.021; multiple comparisons found statistically significant difference(<i>P</i><0.05). Celecoxib had raised the expression of Cx43 mRNA in diabetic rats with real time quantitative PCR. Relative value was 0.635±0.084, 0.110±0.061, 0.367±0.074, multiple comparisons found statistically significant difference(<i>P</i><0.05).<p>CONCLUSION: Expression of Cx43 decreases in retina of SD rats with DM. Celecoxib can relieve the decrease of Cx43 expression in retina of them.

Effect of hyperbaric oxygen preconditioning on expression of MMP-2, MMP-9 and Cx43 in rat abdominal skin flap model of ischemia-reperfusion injury / 中华医学美学美容杂志

Objective To study the effect of hyperbaric oxygen preconditioning management on the expression of MMP-2,MMP-9 and Cx43 in a rat abdominal skin flap model of ischemia-reperfusion injury.Methods Eighteen male adult SD rats,weight ranged from 220-250 g,were randomly divided into three groups:sham group (SH),ischemia-reperfusion group (IR) and hyperbaric oxygen preconditioning group (HBO).All the rats in HBO group received hyperbaric oxygen treatment twice each day,apart 12 hours,during the last three days before operation.Abdominal skin flap with superficial epigastric artery as pedicle was established.In HBO and IR group,3 hours of ischemia was performed.On the 3rd postoperative day,samples were taken to assess the expression of MMP-9,MMP-2 and Cx43 by immunohistochemical staining and Western blot.Results Compared with IR group,the expression of Cx43 was significantly increased (IR 15.03±3.66;HBO 36.01±4.12) and the ex pression of MMP-9 and MMP-2 was decreased (MMP 2:IR 12.01±1.23;HBO 5.98±1.48;MMP9:IR 16.77±2.01;HBO 11.48±1.77).Conclusions Hyperbaric oxygen preconditioning for the rat abdominal skin flap model of ischemia-reperfusion injury has inhibitory effect on the expression of MMP-9 and MMP-2,and stimulative effect on the expression of Cx43.

Effect of Cx43 on proliferation of C6 glioma cells and its mechanisms / 中国药理学通报

Aim To explore the effect of Cx43 over-expression on proliferation of C6 cells and its mechanisms by transfecting pCMV-Cx43cDNA plasmid into C6 cells.Methods pCMV-Cx43cDNA plasmid was transfected into C6 cells by liposome to up-regulate the expression of Cx43, and C6 cells with over-expression of Cx43 was stably cloned by using G418.Determination of cell doubling time and soft agar colony formation assay to detect the degree of cell proliferation.The cells were treated with ERK1/2 specific blocker PD98059(30 μmol·L-1) and p38MAPK specific blocker SB202190(10 μmol·L-1)respectively, the expression of Cx43, p-Cx43, p-ERK1/2 and p-p38MAPK of each group were detected by Western blot, and the activity of each group was detected by MTT Assay.Results pCMV-Cx43cDNA plasmid was transfected into C6 cells successfully.Cell lines with over-expression Cx43(C6-Cx43) or empty vector (C6-pCMV) were stably selected by using G418.Determination of cell doubling time and soft agar colony formation experiments showed that the proliferative rate and the colony number of C6-Cx43 group were significantly decreased, compared with that of C6 group and C6-pCMV group(P<0.01);ERK1/2, p38MAPK specific blockers were treated with each group,Western blot showed that the expression of Cx43 protein was increased(P<0.01), while p-Cx43 protein was decreased (P<0.05) in C6-Cx43+PD98059 group and C6-Cx43+SB202190 group,compared with that of C6-Cx43 group.Conclusion Cx43 may decrease the proliferation of glioma cells through ERK1/2, p38MAPK pathways.

Effects of estrogen on the vasomotor function of rats' renal interlobar arteries and Cx43 expression after renal ischemia-reperfusion injury / 西安交通大学学报(医学版)

Objective To investigate the effect of estrogen (E2) on the connexin43 (Cx43) expression of renal interlobar arteries after renal ischemia-reperfusion injury (I/R).Methods The experiment was carried out in vivo using an SD rat I/R model.SD rats were randomly divided into normal group,sham-operation group,I/R group,and estrogen-intervention group.The functional changes of the kidney were analyzed after 24 hours of I/R;nephridial tissue section was stained by hematoxylin-eosin (HE),and Paller scores were used to evaluate the degree of kidney damage.Pressure myography was utilized to detect the vasomotor function of renal interlobar arteries.Immunofluorescence technique,qRT-PCR and Western blot were applied to determine the expression of Cx43 in renal interlobar arteries in different groups.Results Estrogen markedly decreased the levels of Cr and BUN in the serum of I/R rats (P＜0.05),and the damage of the kidney tissue could be improved noticeably.The vasomotor rate of renal interlobar arteries was (24.80 ± 3.70)％ after I/R and (41.60 ± 3.50)％ after treatment with estrogen,which was higher than that of I/R group (P＜0.05).The expression of Cx43 was lower in renal interlobar arteries of estrogen-intervention group than that in I/R group (P＜0.01).Conclusion Estrogen may reduce vascular tension and boost dilation of the artery by inhibiting Cx43 expression and GJ function.Therefore,estrogen may attenuate the damage of I/R and improve renal function.

Role of gap junction Cx43 in dexmedetomidine for prevention of reperfusion induced arrhythmias during ischemia-reperfusion in isolated rabbit hearts / 临床麻醉学杂志

Objective To investigate the effect of dexmedetomidine on myocardial repolarization heterogeneity and the expression of Cx43 during ischemia-reperfusion and the role of Cx43 in the dexmedetomidine for inhibition of myocardial repolarization heterogeneity during ischemia-reperfusion in isolated rabbit hearts.Methods Eighteen healthy adult rabbits,weighing (2.0±0.5) kg,were randomly divided into three groups after Langendorff isolated heart perfusion model had been prepared and K-H fluid had been perfused and balanced 15 min.In the control group (group C),37℃ K-H fluid was continuously perfused and balanced for 150 min.In group IR,K-H fluid was stopped after perfusion continue filling for 15 min,and then made the cardiac stop for 60 min with the injection of Thomas solution 10 ml/kg while the heart was protected by the 4℃ Thomas solution around.Following the reperfusion of 4℃ Thomas solution 5 ml/kg was performed for 30 min and the heart was resuscitated by the perfusion of K-H fluid for 60 min.In dexmedetomidine group given (group DEX),dexmedetomidine was added in the K-H fluid and the Thomas solution 25 ng/ml.The other procedures were the same as those of group IR.The heart rate (HR),90% monophasic action potential duration (MAPD90) were recorded at the time of balance perfusion record 15 min (T0),continue perfusion 15 min/balance 30 min (T1),reperfusion 30 min/balance 120 min (T2) and reperfusion 60min/balance 150 min (T3).The transmural dispersion of repolarization (TDR) was calculated.To observe the cardiac reperfusion arrhythmia and rebeating time and recording.Detection expression of Cx43 in the left ventricular myocardial by Western blot and immunohistochemistry at T3.Results Group DEX cardiac resuscitation time was significantly shorter than that of group IR (P<0.05).In group DEX.Compared with T0,HR was significantly decreased and TDR was significantly increased in groups IR and DEX at T2、T3 (P<0.05).Compared with group IR,the TDR of group DEX was significantly decreased at T2、T3 (P<0.05).Compared with group C,the expression of Cx43 was decreased (P<0.05) and the distribution was not uniform in groups IR and DEX.Compared with group IR,the expression of Cx43 was decreased (P<0.05) and the distribution was improved in group DEX.Conclusion Dexmedetomidine could inhibits myocardial repolarization heterogeneity of ischemia-reperfusion injury,and thus play a stable cardiac conduction,reduce reperfusion arrhythmias,and its mechanism may be that dexmedetomidine could inhibits gap junctional uncoupling and inhibits expression and distribution of connexins decreased.

Effects of TLR4?mediated Cx43 expression on proliferation and apoptosis of bladder cancer cells by LPS / 实用医学杂志

Objective To study the impacts of lipopolysaccharide on expressions of Cx43 and TLR4 proteins in bladder cancer cell lines and on cell proliferation and apoptosis. Methods 5637 cells were cultured in vitro. After stimulation with LPS,expressions of Cx43 and TLR4 proteins were detected by Western blot in bladder cancer 5637 cell. The proliferation and apoptosis in the 5637?control group,5637?LPS group,and 5637?LPS +cisplatin group were detected by CCK?8(cell counting kit)and flow cytometry. Results The gene expression of Cx43 in the 5637?LPS group was significantly higher than that in the 5637?control group(t=3.892,P=0.012). The expressions of TLR4 and Cx43 in 5637?LPS group were significantly higher than those in the 5637?control group(t=7.029,P=0.019;and t=18.17,P=0.003). The proliferation was significantly decreased in the 5637?control group as compared with the 5637?LPS group (t = 8.756,P = 0.018). The apoptotic rate was (8.3 ± 1.58)% in the 5637?control group and (7.8 ± 2.03)% in the 5637?LPS group,with no significant statistical difference(t = 2.935,P = 0.099). However,the rate of the 5637?cisplatin group(60 ± 4.35)%was higher than that in 5637?cisplatin + LPS group(52 ± 6.25)%,the difference was statistically significant(t = 6.992,P =0.019). Conclusions Under the stimulation of LPS,bladder tumor cells may induce tumor cells to escape immune surveillance by increasing the expressions of TLR4 and Cx43.

Effects of propofol on the expression of gap junction Cx43 in renal arteryin rats with renal ischemia reperfusion injury / 西安交通大学学报(医学版)

Objective To observe the effects of propofol intervention on renal ischemia-reperfusion injury on the expression of Cx43 in rat renal interlobar artery.Methods Male SD rats were randomly divided into control 4h and 24h groups (control), sham operation 4h and 24h groups (sham), ischemia reperfusion 4h and 24h groups (I/R), propofol 4h and 24h groups (propofol), and fat emulsion 4h and 24h groups (intralipid).Ischemia/reperfusion model was prepared by resection of right kidney and noninvasive arterial occlusion of left kidney, with renal ischemia for 45min and reperfusion for 4h or 24h depending on different group.Serum urea nitrogen (BUN) and creatinine (Cr) were detected by automatic biochemical analyzer.HE staining was used to observe the pathological changes of renal tissue.The changes of renal artery systolic and diastolic lobes were examined by pressure myographic technique.The expression of Cx43 protein in renal interlobar artery was analyzed by Western blot.Results The concentrations of serum BUN and Cr in sham group did not differ significantly from those in the blank control group.Renal HE staining showed no significant lesions;the pressure myogram of motor renal interlobar artery contraction rate showed no significant difference.The expression of Cx43 protein did not change significantly.Compared with sham operation group, the concentrations of serum BUN and Cr in ischemia-reperfusiongroup were significantly increased.HE slices kidney showed that the pathological changes of renal tissue became obvious;pressure motor indicated renal interlobar artery contraction rate was decreased;the expression of Cx43 protein was increased significantly (P<0.05).Compared with ischemia-reperfusion group, the concentrations of serum BUN and Cr in propofol group were decreased;renal HE slices showed reduced renal tissue lesions, increased renal interlobar artery contraction rate, and decreased expression of Cx43 protein (P<0.05).Conclusion Propofol can change renal ischemia-reperfusion injury by reducing the expression of Cx43 protein in vasomotor in renal interlobar artery.

Effects of Ang II perfusion on transmural heterogeneous of Cx43 in acute myocardial ischemia reperfusion

Objective: To observe the effects of angiotensin II(Ang II) perfusion on transmural heterogeneity of Cx43 expression in the rabbit model with acute myocardial ischemia reperfusion (MIR), and investigate the role of rennin-angiotensin system in malignant ventricular arrhythmia induced by MIR. Methods: Twenty rabbits were randomly divided into MIR group (n = 10) and Ang II group (n = 10). MIR model was produced with traditional ligation and opening of the anterior descending coronary artery in all animal. The hearts in vitro in the MIR group and the Ang II group were perfused with simply improved Tyrode's solution and containing Ang II Tyrode's solution respectively. 90% monophasic action potential repolarization duration, transmural dispersion of repolarization, Cx43 protein (Cx43-pro) and mRNA (Cx43-Cq) expression in subepicardial, midmyocardial and subendocardial myocardium were measured in both groups. The greatest differences of Cx43-pro and Cx43-Cq among three myocardial layers were calculated and shown with ΔCx43-pro and ΔCx43-Cq respectively. Results: After Ang II perfusion, 90% monophasic action potential repolarization duration among three myocardial layer were significantly prolonged (P < 0.05 and P < 0.01), and transmural dispersion of repolarization also significantly increased compared with the MIR group (P < 0.05). Compare with the MIR group, three myocardial Cx43-pro and Cx43-Cq expression in the Ang II group were significantly decreased (P < 0.05 and P < 0.01), but ΔCx43-pro and ΔCx43-Cq were significant increased. Conclusions: Renin-angiotensin system increases transmural heterogeneity of Cx43 expression in the rabbit model with MIR by Ang II, and enlarge transmural dispersion of repolarization among three myocardial layers of left ventricular which induces malignant ventricular arrhythmia.