| Objective:Bronchopulmonary dysplasia(BPD)is a common chronic lung disease in very low birth weight infant and extremely low birth weight infant.The incidence rate of BPD is high in the worldwide,seriously affecting the prognosis of premature infants.Therefore,the diagnosis and prevention of BPD has become a research hotspot in neonatology.Alveolar maldevelopment is a key pathological feature of BPD.Aberrant alveolarization results in pulmonary dysfunction in BPD infants,even lasting into adulthood.Oxidative stress is a key pathogenesis of BPD.Prenatal and postpartum infection,unavoidable oxygen inhalation,mechanical ventilation and other adverse factors could trigger or aggravate the oxidative stress response of lung tissue,blocking alveolar development of premature infants.Therefore,it is necessary to explore the pathological mechanism of alveolar maldevelopment induced by oxidative stress.Type II alveolar epithelial cell(ATII)has stem cell characteristics,which can not only self renew,but also transdifferentiate into type I alveolar epithelial cell(ATI).ATII is the key target of BPD alveolar maldevelopment.Oxidative stress–induced excessive apoptosis of ATII is the crucial mechanism in alveolar maldevelopment of BPD.Mitogen activated protein kinase(MAPK)pathway is the most frequently mentioned signal pathway for regulating cell stress.MAPK pathway includes MKKK,MKK and MAPK tertiary kinases.Apoptosis signal-regulated kinase 1(ASK1)mediated apoptosis is involved in the pathogenesis of several oxidative stress–related diseases such as brain ischemia,ischemic heart disease,and Alzheimer’s disease.Under oxidative stress conditions,ASK1 resulted in apoptosis by activating pro-apoptotic c-Jun NH2-terminal kinase(JNK)and p38 mitogen–activated protein kinase(MAPK)pathway.Previous studies indicated JNK and p38 MAPK pathway had been most frequently reported to have roles in the induction of apoptotic responses in hyperoxia lung injuries.Normalfall,the organisms mobilize the intercellular communication network to dilute the cytotoxicity of some damaged cells,which is conducive to the survival of damaged cells(the "Good Samaritan" effect).When cellular stress is severe or sustained,intercellular communication networks expand cytotoxicity,inducing more cells injury or even death(the bystander effect).Gap junction channels(GJCs)are transmembrane channels,providing one of the most common forms of intercellular communication.GJCs may also be hazardous by providing a channel to spread cytotoxicity to the adjacent cells,and amplify cell injury.GJCs blockers inhibit the intercellular transmission of stress signals to maintain cellular homeostasis.GJCs are formed by paired connexons,which are present on the membranes of adjacent cells.Each connexon is made up of six connexin(Cx)subunits.Cx26,Cx32,Cx43,and Cx46 express in alveolar epithelial cells.Connexin 43(Cx43)is the most ubiquitous and critical GJ protein in alveolar epithelial cells.Based on the above,this study first explored the expression and localization of Cx26,Cx32,Cx43 and Cx46 in vivo.Secondly,we explored whether Gap26(Cx43-GJCs blocker)regulates oxidative stress,apoptosis,and alveolar development in vivo.Finally,we explored the potential regulatory mechanism between Cx43 and oxidative stress and apoptosis in vitro.Methods:1.In vivo,the expression and localization of Cx26,Cx32,Cx43 and Cx46 in the lung tissue of neonatal rats with BPD were determined.(1)Animal models and groups:According to our established procedure,neonatal rats were exposed to 21% O2(normoxia)or 85% O2(hyperoxia)from postnatal day(PN)1 to 14.(2)Experimental method:The lung paraffifin sections were stained with hematoxylin and eosin(HE)for examination of the lung architecture.And alveolar development was assessed by counting radiative alveolar counting(RAC)values.Lung tissue was examined by TUNEL staining.The ROS,MDA and GSH levels were respectively determined by ROS,MDA and GSH kits.The protein relative expression of cleaved caspase-3 and caspase-3 were measured.The expression and localization of Cx26,Cx32,Cx43 and Cx46 in alveoli were detected by immunohistochemistry and immunofluorescence double staining.The total and membrane protein expression of Cx26,Cx32,Cx43,and Cx46 were determined by Western blot.The m RNA expressions of Cx26,Cx32,Cx43 and Cx46 were detected by Real-time PCR.2.In vivo,we detedcted the effects of Gap26(Cx43-GJCs blocker)on alveolar development,oxidative stress,apoptosis and the activity of ASK1-JNK/p38 pathway.(1)Animal models and treatment:Gap 26 was a specific Cx43 mimic peptide.Gap 26,as a kind of Cx43-GJCs inhibitor,blocked Cx43-medicated cell-cell coupling.The neonatal rats were exposed to 21% O2(normoxia)or 85% O2(hyperoxia)from postnatal day(PN)1 to 14.The neonatal rats were randomly divided into 4 groups: normoxia+saline group,normoxia+Gap26 group,hyperoxia+saline group and hyperoxia+Gap26 group.Some neonatal rats were treated with Gap26 via intraperitoneal injection at 50 μg/kg body weight once daily from PN1 d to PN14 d,and some were treated with the same amount of sterile saline.(2)Experimental method:Lung histomorphological changes were visualized by HE staining,and alveolar development was assessed by counting RAC values.The ROS,MDA,and GSH levels were respectively determined by ROS,MDA,and GSH kits.Lung tissue was assessed by TUNEL staining.The protein expression of p-ASK1/ASK1,p-JNK/JNK,p-p38/p38 and cleaved caspase-3/caspase-3 were measured by Western blot.Cx43 expression was determined by immunofluorescence.We detected Cx43 protein expression by Western blot and m RNA expression by Real-time PCR.3.In vitro,we explored the potential mechanism between Cx43 and oxidative stress and apoptosis.(1)Cell culture and treatment:RLE-6TN cells were type II alveolar epithelial cells of rats.RLE-6TN cells were exposed under 21% O2 or 85% O2.The RLE-6TN cells were randomly divided into 5groups,including normoxia group,normoxia+Gap26 group,hyperoxia group,hyperoxia+Gap26 group and hyperoxia +NAC group.In the NAC(ROS scavenger)treatment experiments,RLE-6TN cells were treated with 10 m M NAC.In the Gap26 treatment experiments,we treated RLE-6TN cells with 150 μM Gap26.(2)Experimental method:Intercellular communication was detected by dye-coupling assay.We detectedthe ROS level of RLE-6TN cells by dichlorodihydrofluorescein diacetate(DCFH-DA)probe.The apoptosis levels were determined by Annexin V-FITC/Propidium Iodide double-dye kit.The activity of ASK1-JNK-p38 pathway was measured by Western blot.The protein expression ratio of cleaved caspase-3/caspase-3 were measured.Cx43 expression and location were detected by immunofluorescence,Western blot and Real-time PCR.Results:Part 1:1.Hyperoxia exposure arrested alveolar development in newborn rats.The neonatal rats exposed to 21% O2 had a standard distal lung architecture with well-formed alveoli and a normal RAC value.However,distal lung histology in neonatal rats exposed to 85% O2 showed simplifcation of the distal lung architecture,with fewer,larger alveoli and a lower RAC value(P < 0.01).2.Hyperoxia exposureincreased oxidative stress in the lung tissue of neonatal rats.Hyperoxia exposure increased ROS and MDA production(P < 0.01),and decreased GSH production(P < 0.01).3.Hyperoxia exposure increased apoptosis in the lung tissue of neonatal rats.AI and cleaved caspase-3/caspase-3 ratio in H group were higher than those in N group(P < 0.01).4.Expression and localization of Cx26,Cx32,Cx43 and Cx46 in lung tissue of BPD rats.(1)Immunohistochemical results showed that Cx26,Cx32,Cx43 and Cx46 were all seen in normoxia group,Cx43 was significantly increased in hyperoxia group,Cx26 was slightly increased in hyperoxia group,and there was no significant difference in the expression of Cx32 and Cx46 between the two groups.(2)Immunofluorescence double staining results indicated that Cx26 was expressed in ATI and ATI,mainly in ATII;Cx32 was only expressed in ATII cells;Cx43 is expressed in both ATI and ATII;Cx46 was expressed in ATI and ATI,mainly in ATI.(3)The results of Real-time PCR showed that the expression levels of Cx26,Cx32,Cx43 and Cx46 genes in hyperoxia group were significantly higher than those in normoxia group(P < 0.01);(4)Cx26 membrane protein and total protein expression in hyperoxia group were increased significantly at PN7 d and PN14d(P < 0.01);Cx32 membrane protein expression in hyperoxia group was increased significantly at PN7 d and PN14d(P <0.01),but Cx32 total protein expression in hyperoxia group was not changed;Cx43total protein expression was higher significantly at PN7 d and PN14 d with 85% O2(P< 0.01),while Cx43 membrane protein expression was increased from PN1 d with85% O2(P < 0.01),and this increasing trend continued until PN14d(P < 0.01);there was no difference in Cx46 total protein and membrane protein expression between hyperoxia group and normoxia group.5.Correlation analysis among Cx43 membrane protein expression,ROS levels,AI and RAC value.Cx43 membrane protein expression was positively associated with ROS level(Pearson analysis,P < 0.01).Cx43 membrane protein expression was positively associated with AI(Pearson analysis,P < 0.01).Cx43 membrane protein expression was negatively associated with RAC value(Pearson analysis,P < 0.01).Part 2:1.Gap26 improved alveolar development of rats with hyperoxia exposure.HE staining results showed that the hyperoxia exposed and Gap26-treated rats had increased alveolarization with a higher RAC value compared to the rats with hyperoxia exposure(P < 0.01).2.Gap26 treatment alleviated oxidative stress of neonatal rats with hyperoxia exposure.Gap26 treatment downregulated the ROS level and MDA level of neonatal rats with hyperoxia exposure(P < 0.01).The hyperoxia exposed and Gap26-treated rats had higher GSH level compared to the rats with hyperoxia exposure(P < 0.01).3.Gap26 treatment decreased apoptosis of neonatal rats with hyperoxia exposure.TUNEL staining results showed that Gap26 treatment decreased AI of rats exposed to hyperoxia(P < 0.01).Treatment with Gap26 reversed the increase in the cleaved caspase-3/caspase-3 ratio under hyperoxia conditions(P < 0.01).4.Gap26 treatment inhibited ASK1-JNK/p38 signaling pathway.Gap26 treatment reduced the activity of the ASK1-JNK/p38 signaling pathway in rats exposed to hyperoxia(P < 0.01).5.Gap26 treatment decreased Cx43 expression of neonatal rats with hyperoxia exposure.(1)Gap26 treatment decreased the Cx43 m RNA expression of neonatal rats exposed to hyperoxia(P < 0.01).(2)Immunofluorescence results suggest that compared to the rats with hyperoxia exposure,hyperoxia-exposed and Gap26-treated rats had lower fluorescence intensity of Cx43.(3)Gap26 treatment decreased the Cx43 total protein and membrane protein expression of neonatal rats exposed to hyperoxia(P < 0.01).Part 3:1.In ATII cells with hyperoxia exposure,the expression of Cx43 was up-regulated,and the ability of intercellular communication was enhanced.Hyperoxia exposure increased Cx43 m RNA,total protein and membrane protein expression in RLE-6TN cells(P < 0.01).To assess the capacity of intercellular communication,we subjected RLE-6TN cells to the dye coupling assay.We found that compared with normoxia exposure,hyperoxia exposure resulted in increased diffusion capacity of fluorescent dye between cells(P < 0.01).2.NAC treatment significantly downregulated Cx43 expression and intercellular communication capacity and level of apoptosis of RLE-6TN cells in a hyperoxic environment.(1)ROS levels were measured by flow cytometry,and the results indicated that NAC downregulated the ROS level of RLE-6TN cells in a hyperoxic environment(P< 0.01).(2)As expected,NAC treatment decreased the Cx43 m RNA,total protein and membrane protein expression of RLE-6TN cells exposed to hyperoxia(P < 0.01).NAC treatment weakened diffusion capacity of fluorescent dye between cells with hyperoxia exposure(P < 0.01).(3)The apoptotic level was measured by flow cytometry,and the results suggested that NAC decreased the apoptotic rate of RLE-6TN cells with hyperoxia exposure(P < 0.01).3.Gap26 treatment alleviated oxidative stress,thus decreasing apoptosis via inhibiting ASK1-JNK/p38 signaling in ATII cells with hyperoxia exposure.(1)Gap26 treatment significantly weakened the ability of intercellular communication(P < 0.01).(2)Gap26 decreased ROS production of RLE-6TN in hyperoxia exposure(P <0.01).(3)RLE-6TN cells exposed to hyperoxia had increased phosphorylated protein expression of ASK1,JNK1/2,and p38 MAPK,while Gap26 treatment significantly reversed these changes(P < 0.01).(4)Gap26 decreased the cleaved caspase-3/caspase-3 ratio of RLE-6TN cells in hyperoxia exposure(P < 0.01).The apoptotic level was measured by flow cytometry,and the results suggested that Gap26 decreased the apoptotic rate of RLE-6TN cells in hyperoxia exposure(P < 0.01).4.Gap26 downregulated Cx43 expression of ATII cells with hyperoxia exposure.(1)Immunofluorescence results indicated that Gap26 treatment downregulated Cx43 fluorescence intensity of RLE-6TN cells in hyperoxia exposure.(2)Gap26 treatment decreased the Cx43 m RNA expression of RLE-6TN cells exposed to hyperoxia(P < 0.01).(3)Gap26 treatment decreased the Cx43 total protein and membrane protein expression of RLE-6TN cells exposed to hyperoxia(P < 0.01).Conclusions:1.At the m RNA level of lung tissue,the expressions of Cx26,Cx32,Cx43 and Cx46 were increased in BPD rats;at the total protein level,the expression of Cx26 and Cx43 in BPD rats were increased;at the membrane protein level,the expression of Cx26,Cx32 and Cx43 in BPD rats were increased.Cx43 was the first high expression in the membrane protein level and lasted the longest.2.Gap26 as Cx43-GJCs blocker inhibited ASK1-JNK/p38 signal pathway,down-regulated oxidative stress and apoptosis,and improved alveolar development of neonatal rats with hyperoxia exposure.3.In ATII cells with hyperoxia exposure,the expression of Cx43 was up-regulated and the ability of intercellular communication was enhanced;Cx43-GJCs blocker Gap26 inhibited intercellular communication,decreased ASK1-JNK/p38 signal pathway,downregulated ROS level and apoptosis.4.ROS up-regulated Cx43 expression;Gap26 downregulated ROS level,in turn dereased Cx43 expression.There may be an interactive regulation relationship between ROS and Cx43. |
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