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Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

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International Journal of Ophthalmology(English edition) 2021年第3期14卷 341-348页

作者： Xiao-Shuo Zheng Hui Zheng Dan Xu Ping-Ping Liu Bing Li Zi-Mu Cao Yang Liu Ye Liu Department of Ophthalmology the Fith Affiliated HospitalSun Yat-sen UniversityZhuhai 5190001 Guangzhou ProvinceChina Institute of Enironmental Systems Biology Environmental Science and Enginecring CollegeDalian Maritime UniversityDalian 116027Liaoning ProvinceChina Department of Pathology the Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000Guangzhou ProvinceChina

AIM:To study the effect of zymosan,a ligand found on the surface of fungi,on gap junctional intercellular communication(GJIC)in cultured human corneal fibroblasts(HCFs).METHODS:Zymosan was added to the medium of cultured HCFs with or without the administration of mitogenactivated protein kinase(MAPK)inhibitors or the inhibitor kappa B kinase 2(IKK2)inhibitor *** protein and m RNA levels of connexin 43(Cx43)in HCFs were measured by Western blot,immunofluorescence,and quantitative reverse transcription-polymerase chain reaction(q RT-PCR)*** GJIC activity was tested using a dye-coupling ***:The reduction of Cx43 protein and m RNA levels as well as a significant decrease in GJIC activity were observed in cultured HCFs when zymosan was added into the culture *** with controls(no zymosan),the protein level of Cx43 was reduced by 45%and 54%in the presence of zymosan at 200 and 600μg/m L,respectively(P43 was reduced by 98%in the presence of zymosan(P43 expression and GJIC activity were attenuated by the administration of PD98059[an extracellular signal-regulated kinase(ERK)signaling inhibitor](P43 expression and GJIC activity in HCFs may induce damage of corneal stroma during corneal fungal infection.

关键词： corneal fibroblast zymosan gap junction connexin 43 MAPKs

THE EFFECT OF ALL-TRANS RETINOIC ACID ON GAP JUNCTIONAL INTERCELLULARCOMMUNICATION AND connexin 43 GENE EXPRESSION IN GLIOMA CELLS

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Chinese Medical Sciences Journal 2002年第1期17卷 22-26页

作者：张雪峰任祖渊左瑾苏长保王任直常永生方福德 Neurosurgery Department Peking Union Medical College Hospital CAMS & PUMC Beijing 100730

To illuminate the regulating effect of all trans retinoic acid (ATRA ) on gap junctional intercellular communication (GJIC) and connexin 43 (Cx43) ge ne expression in glioma cells, which is tissue and organ specific. Method. Rat C6 glioma cells were exposed to ATRA at a concentration of 1, 10, 10 0 μmol/L respectively, and the GJIC function of the cells was examined with scr ape loading dye transfer assay 24 hours, 48 hours and 72 hours after ATRA treat ment. The effect of ATRA on Cx43 gene expression was measured with semiquantitat ive reverse transcription polymerase chain reaction (RT PCR) 24 hours after ATR A exposure. Results. The GJIC function of C6 glioma cells was significantly increased by ATR A at each concentration applied. The dye passed 4 to 5 rows of cells from the sc raping edge in ATRA treated cells, but only 1 or 2 rows in the control. The augm ent effect was observed 24 hours after each concentration ATRA treatment, and la sted till 72 hours after treatment with 1μmol/L and 10μmol/L ATRA. Forty eigh t hours after exposed to 100μmol/L ATRA, the enhancement of GJIC was less obvi ous. There was no significant increase induced by ATRA on the transcription of C x43 gene, as demonstrated by semiquantitative RT PCR. Conclusion. ATRA turned out to be a potent enhancer on GJIC function in C6 gliom a cells, and the enhancement effect was most probable at post transcriptional l evel.

关键词： all trans retinoic acid gap junctional intercellular communication connexin 43 glioma

connexin 43 remodeling induced by LMNA gene mutation Glu82Lys in familial dilated cardiomyopathy with atrial ventricular block

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Chinese Medical Journal 2010年第8期123卷 1058-1062页

作者： SUN Li-ping WANG Lin WANG Hui ZHANG Yin-hui PU Jie-lin Cardiovascular Institute and Fu Wai Hospital Chinese Academy of Medical Sciences Peking Union Medical College Beijing 100037 China

Background Mutations in the lamin A/C gene （LMNA） may cause familial dilated cardiomyopathy （dilated cardiomyopathy） characterized by early onset atrio-ventricular block （A-V block） before the manifestation of dilated cardiomyopathy and high risk of sudden death due to ventricular arrhythmia, which is very similar to the phenotype of gap junction related heart disease. This study aimed to determine the expression and localization of connexins in neonatal myocytes transfected with wild-type （WT） or mutant LMNA to elucidate how these mutations cause heart diseases. Methods We studied the connexin 43 （Cx43） and connexin 40 （Cx40） expression in cultured neonatal myocytes transfected with wild-type （WT） or mutant LMNA （Glu82Lys （E82K） and Arg644Cys （R644C）） using confocal imaging and Western blotting analysis. Results Cx43 protein expression was reduced by 40% in cells transfected with LMNA E82K than that in cells transfected with WT LMNA cDNA. Confocal imaging showed that the Cx43 located inside the cells by LMNA E82K. By contrast, LMNA E82K mutation had no effect on expression and localization of Cx40. LMNA R644C transfection did not show any significant effects on gap junctions at all. Conclusions Our findings suggest that LMNA E82K significantly reduced the Cx43 expression and altered its localization which may be one of the pathological mechanisms underlying LMNA-related heart disease.

关键词： lamin A/C LMNA mutations connexin 43 atrio-ventricular block

connexin 43 co-locolizes and regulates the L type calcium channel current in atrial myocytes

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South China Journal of Cardiology 2015年第2期16卷 114-121页

作者：饶芳薛玉梅邓春玉余细勇肖定璋陈少贤林秋雄杨慧邝素娟刘晓颖朱杰宁吴书林 Department of Cardiology Guangdong Cardiovascular Institute Research Center of Medical Sciences Guangdong General Hospital Guangdong Academy of Medical Sciences

Background Atrial fibrillation （AF） is the most common sustained cardiac arrhythmia without effective treatment. AF is associated with atrial conduction disturbances caused by electrical and / or structural remodeling. But the role of connexin （Cx） 43 in the regulation of L type calcium channel （LCC） remains unclear. We hypothesized that Cx 43 might co-localize and regulate the L type calcium channel current （ICa, D. Methods Real-time PCR and whole-cell patch clamp were used to detect the expression of LCC lc subunit and the cur rent density of Ica, L, before and after Cx 43 knocking down respectively. The co-localization of Cx 43 with LCC was investigated by co-immunoprecipitation and confocal microscopy. Results Knocking down of Cx43 significantly inhibited the current density of ICa, L through decreasing the gene expression of LCC alc in cul tured atrium-derived myocytes （HL-1 cells）. Cx43 co-localized with LCC eric subunit in atrial myocytes. Conclusions Cx 43 regulates the ICa, L in atrial myoctyes through LCC, representing a potential pathogenic mechanism in atrial arrhythmias.

关键词： connexin 43 L type calcium channel current HL-1 cells atrial fibrillation

connexin 43 gene-modified BMSCs for treating myocardial infarction

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South China Journal of Cardiology 2011年第2期12卷 112-117页

作者：李晓红江雪燕朱杰宁麦丽萍肖定璋符永恒邝素娟张光峰余细勇 Medical Research Center Guangdong General HospitalGuangdong Provincial Cardiovascular Institute Department of Rheumatology and Immunology Guangdong General HospitalGuangdong Academy of Medical Sciences

Background stem cells （B infarction （MI controversial. Stem cells have MSCs） derived ）. The in vivo c Poor moderate multilineage differentiation capacity, which makes bone marrow mesenchymal relatively easy and provides a promising alternative treatment for myocardial ardiac differentiation and functional effects of unmodified BMSCs after MI are survival benefits of BMSCs-implanted rats were caused by incomplete electromechanical integration induced by infarcted myocardium. This study was to therapeutic efficacy in MI. Methods tissue heterogeneity between myocytes and engrafted BMSCs in the investigate whether connexin43 （Cx43） modified BMSCs improve Lentivirus mediated Cx43 gene was transfeeted into human BMSCs. Flowcytometry was used to detect cell surface markers of BMSCs before and after Cx43 transfection. Cx43 expression levels were detected by fluorescent microscope and western blotting. Post-MI intramyocardial injection of 1 x 106 stem ceils was compared with injection of medium alone echocardiography. Results Heart function was detected by Cx43 of BMSCs could be effectively overexpressed by lentivirus mediated gene transfection. Flowcytometry showed that after Cx43 overexpression, the surface markers of BMSCs were greatly decreased. Immunofluorescent microscopy revealed GFP-posotive transplanted BMSCs among the MI area. The Cx43-BMSCs-treated heart, assessed by echocardiography, improved ejection fraction compared with PBS- and mock-BMSCs-treated hearts （P 〈 0.01）. Conclusions Lenti-Cx43 can be effectively transfected into BMSCs. Cx43 may act as a potential target for improving the therapeutic efficacy of BMSCs in ischaemic heart disease.

关键词： connexin 43 stem cell myocardial infarction transplantation

Mechanism of Regulatory Effect of MicroRNA-206 on connexin 43 in Distant Metastasis of Breast Cancer

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Chinese Medical Journal 2016年第4期129卷 424-434页

作者： Zi-Jing Lin Jia Ming Lu Yang Jun-Ze Du Ning Wang Hao-Jun Luo Department of Breast Thyroid and Pancreas Surgery Second Affiliated Hospital of Chongqing Medical University Chongqing 400010 China Breast Disease Center Southwest Hospital Affiliated to Third Military Medical University Chongqing 400038 China

Background： MicroRNA-206 （miR-206） and connexin 43 （Cx43） are related with the distant metastasis of breast *** remains unclear whether the regulatory effect of miR-206 on Cx43 is involved in metastasis of breast ***： Using quantitative real-time polymerase chain reaction and Western blot, the expressions of miR-206 and Cx43 were determined in breast cancer tissues, hepatic and pulmonary metastasis （PM）, and cell lines （MCF-10A, MCF-7, and MDA-MB-231）.MCF-7/MDA-M-231 cells were transfected with lentivirus-shRNA vectors to enhance/inhibit miR-206, and then Cx43 expression was *** counting kit-8 assay and Transwell method were used to detect their changes in proliferation, migration, and invasion *** mutant plasmids of Cx43-3＆#39; untranslated region （3＆#39;UTR） at position 478-484 and position 1609-1615 were *** reporter assay was performed to observe the effects of miR-206 on luciferase expression of different mutant plasmids and to confirm the potential binding sites of ***： Cx43 protein expression in hepatic and PM was significantly higher than that in the primary tumor, while no significant difference was showed in messenger RNA （mRNA） ***-206 mRNA expression in hepatic and PM was significantly lower than that in the primary ***43 mRNA and protein levels, as well as cell proliferation, migration, and invasion capabilities, were all significantly improved in MDA-MB-231 cells after reducing miR-206 expression but decreased in MCF-7 cells after elevating miR-206 expression, which demonstrated a significantly negative correlation between miR-206 and Cx43 expression （P =0.03）.MiR-206 can drastically decrease Cx43 expression of MCF-7 cells but exerts no effects on Cx43 expression in 293 cells transfected with the Cx43 coding region but the lack of Cx43-3＆#39;UTR, suggesting that Cx43-3＆#39;UTR may be the key in Cx43 regulated by *** expression showed that the inhibition effic

关键词： Breast Cancer connexin 43 Metastasis MicroRNA-206

Occludin and connexin 43 expression contribute to the pathogenesis of traumatic brain edema

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Neural Regeneration Research 2013年第29期8卷 2703-2712页

作者： Wanyin Ren Guojie Jing Qin Shen Xiaoteng Yao Yingchao Jing Feng Lin Weidong Pan Guangdong Medical College Huizhou First People's Hospital

The experimental model of traumatic brain injury was established in Sprague-Dawley rats according to Feeney＇s free falling method. The brains were harvested at 2, 6 and 24 hours, and at 3 and 5 days after injury. Changes in brain water content were determined using the wet and dry weights. Our results showed that water content of tissue significantly increased after traumatic brain injury, and reached minimum at 24 hours. Hematoxylin-eosin staining revealed pathological impairment of brain tissue at each time point after injury, particularly at 3 days, with nerve cell edema, degenera- tion, and necrosis observed, and the apoptotic rate significantly increased. Immunohistochemistry and western blot analysis revealed that the expression of occludin at the injured site gradually de- creased as injury time advanced and reached a minimum at 3 days after injury; the expression of connexin 43 gradually increased as injury time advanced and reached a peak at 24 hours after in-jury. The experimental findings indicate that changes in occludin and connexin 43 expression were consistent with the development of brain edema, and may reflect the pathogenesis of brain injury.

关键词： neural regeneration traumatic brain injury brain edema connexin 43 occludin nerve cells pa-thology tight junction grants-supported paper neuroregeneration

Yimu San improves obstetric ability of pregnant mice by increasing serum oxytocin levels and connexin 43 expression in uterine smooth muscle

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2017年第11期18卷 986-993页

作者： Qi-huan WANG Shuang ZHANG Li-meng QIN Wen-jun ZHANG Feng-hua LIU Jian-qin XU Yun-fei MA Ke-dao TENG College of Veterinary Medicine China Agricultural University College of Animal Science and Technology Beijing University of Agriculture

Prolonged farrowing remains one of the critical challenges in intensive pig farming. This study aims to explore the effects and mechanism of Yimu San （YMS）, a Chinese veterinary medicine micro mist, on delivery ability with mouse models. Thirty-two pregnant mice were randomly divided into a control group and Iow-YMS, med-YMS, and high-YMS groups. The labor process time and stillbirth rate were recorded, the levels of serum oxytocin and prostaglandin E2 （PGE2） were measured with enzyme-linked immunosorbent assay （ELISA）. Contractility measure- ments of the isolated uterus and the expression of connexin 43 （Cx43） in uterine smooth muscle were evaluated. The results showed that compared with the control group, the birth process time and stillbirth rate in the med-YMS and high-YMS groups were remarkably lower. The in vitro uterine contractions, levels of oxytocin, PGE2, and Cx43 in the med-YMS and high-YMS groups were significantly higher than those in the control group. The differences of the above measurements between the Iow-YMS group and the control group were not obvious. It can be speculated that YMS could significantly promote labor in pregnant mice by enhancing the levels of oxytocin, Cx43, and PGE2.

关键词： Yimu San Delivery ability Uterine contraction Oxytocin Prostaglandin 52 connexin 43

Involvement of connexin 43 in acupuncture analgesia

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Chinese Medical Journal 2009年第1期122卷 54-60页

作者： HUANG Guang-ying ZHENG Cui-hong YU Wei-chang TIAN Dai-shi WANG Wei Institute of Integrated Traditional and Western Medicine Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Hubei 430030 China Department of Neurology Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Hubei 430030 China

Background connexin 43 （Cx43） is one of the major components of human keratinocyte gap junctions. To study whether gap junctional intercellular communication participates in the transfer of acupoint signals and acupuncture analgesia, the expression of Cx43 was studied in Zusanli （ST36） acupoints compared with control non-acupoint regions in rats after acupuncture. In addition, Cx43 heterozygous gene knockout mice were used to further explore the relationship between Cx43 and acupuncture analgesia.Methods The expression of Cx43 was detected by immunohistochemistry, immunoblotting, and RT-PCR for the Cx43 protein and mRNA. The influence of the Cx43 gene knockout on acupuncture analgesia was measured by a hot plate and observing the writhing response on Cx43 heterozygous gene knockout mice. Results Immunohistochemistry showed abundant Cx43 expression in some cells in the skin and subcutaneous tissue of rat ST36 acupoints. The mRNA and protein levels of Cx43 in acupoints were significantly higher than those in the control points in the non-acupuncture group, and even more so after acupuncture. The hot plate and writhing response experiments showed that partial knockout of the Cx43 gene decreased acupuncture analgesia. Conclusion Cx43 expression and acupuncture analgesia showed a positive correlation.

关键词： connexin 43 gene knockout acupuncture analgesia hot plate test writhing response