Study On The Role Of Cx43 In Oxidative Damage Of Periodontal Tissues Through Regulation Of JNK/NF-κB Signaling Pathway

Background:Periodontitis is a chronic inflammatory disease that results from infection by periodontal pathogenic microorganisms and dysregulation of the host immune response.It has been found that the oxidative stress microenvironment is one of the key pathogenic mechanisms of periodontitis.Intense oxidative stress can cause irreversible damage to periodontal tissues by producing numerous reactive oxygen species（ROS）.However,the exact role of oxidative stress in the pathogenesis of periodontitis is still unclear.Connexin 43（Cx43）-mediated gap junctions（GJs）（Cx43-GJs）are one of the key structures for maintaining the homeostasis of the internal environment in organisms.In contrast,under pathological conditions,Cx43-GJs allow stress signals such as ROS to transmit among cells,and therefore it is considered to be the biological basis for the deterioration and amplification of tissue damage.Numerous studies have indicated that increased function of Cx43-GJs may be a common feature of diseases in which oxidative stress is the main pathogenic mechanism.However,the role of Cx43-GJs in periodontitis remains poorly understood.The C-Jun N-terminal kinase（JNK）/nuclear factor kappa-B（NF-κB）signaling pathway plays a central role in the regulation of many cellular functions.When this signaling pathway is over-activated,it can lead to disruption of the environment in the organism.However,the regulatory relationship between Cx43-GJs and the JNK/NF-κB signaling pathway and the role in the progression of periodontitis remains to be fully elucidated.Objective:This study aimed to investigate the immunomodulatory role of Cx43-GJs in the pathogenesis of periodontitis,as well as to investigate the mechanisms of oxidative stress,Cx43,and JNK/NF-κB signaling pathways in the progression of periodontitis.Methods:1.In vitro experiments:human periodontal ligament cells（hPDLCs）were routinely isolated and cultured.The hPDLCs were stimulated by hydrogen peroxide（H₂O₂）to construct an in vitro periodontitis oxidative stress microenvironment.The cells were also treated with 18-α-glycyrrhizic acid（GA）（a classical pharmacological inhibitor of gap junction）,and SP600125（an inhibitor of JNK signaling molecules）,for the intervention of cellular experiments.（1）The effects of H₂O₂,GA and SP600125 on the cell viability of hPDLCs were detected by CCK-8 assay.（2）Evaluation of the intercellular communication ability of hPDLCs by"parachute"dye-coupling assay.（3）Intracellular oxidative stress levels were evaluated by measuring ROS content,superoxide dismutase（SOD）activity,and malondialdehyde（MDA）levels.（4）The expression levels of Cx43,P-JNK,NF-κB,apoptosis-related factors,and pro-inflammatory cytokines in hPDLCs were detected by Western blot and Polymerase Chain Reaction（PCR）.2.In vivo experiment:24 male Wister rats were divided into three groups:control group,periodontitis group,and periodontitis+GA intervention group（administration method and dose:gavage administration,30mg/kg/day）,8 rats in each group.Periodontitis models were constructed using the ligation method on the bilateral maxillary first molars of the rats in the periodontitis group and the periodontitis+GA intervention group.2 weeks later,the rats were euthanized and examined for the following indices.（1）The extent of periodontitis was assessed by examining periodontal clinical indicators,Micro-CT analysis,H&E,and TRAP-stained periodontal tissues in rats.（2）The expression levels of the Cx43/JNK/NF-κB signaling pathway in periodontal tissues of rats in each group were detected by Western blot,immunohistochemistry（IHC）,and PCR.（3）The level of oxidative stress in periodontal tissues was evaluated by detecting mitochondrial morphology,mitochondrial membrane potential,ROS content,8-OHd G and SOD1 expression levels in periodontal tissues,and SOD activity and MDA content in serum.（4）The level of apoptosis in periodontal tissues was evaluated by TUNEL staining and detection of the expression levels of apoptosis-related factors.Results:1.Regulation effect of Cx43 on intracellular oxidative stress and inflammatory response.Compared with the Control group,the cell viability of hPDLCs was significantly reduced by H₂O₂ treatment,the expression level of intracellular Cx43,intercellular communication ability,ROS content,and MDA level,as well as the expression level of pro-inflammatory cytokines,were significantly increased,and the antioxidant enzyme SOD activity was decreased;when hPDLCs were pretreated with GA to inhibit the expression and function of Cx43,all the above indicators were reversed.2.The regulatory role of the JNK/NF-κB signaling pathway on intracellular redox status and apoptosis as well as the relationship with the action of Cx43.When hPDLCs were treated with H₂O₂,the intracellular JNK phosphorylation and NF-κB protein expression levels were elevated.While cells were pretreated with SP600125,cell viability of hPDLCs in the H₂O₂+SP600125 group was recovered compared to the H₂O₂ group.In addition,the expression levels of intracellular Cx43,P-JNK,and NF-κB were decreased when cells were pretreated with GA or SP600125compared with the H₂O₂ group,and the expression levels of apoptosis-related factors were also reduced.3.Detection of periodontitis-related indicators in rats.（1）Periodontal clinical index test results.The control group rats failed to probe the periodontal pockets,and the gingival no obvious bleeding,and no looseness of teeth;the periodontitis group rats could probe the deep periodontal pockets,and the gingival bleeding was obvious,and the teeth appeared to different degrees of looseness;the periodontitis+GA group rats had shallow periodontal pockets,and the bleeding degree and looseness of gingival probing were alleviated.（2）Micro-CT scan results.Compared with the control group,the bone quality-related parameters,including BMD,BV/TV,and Tb.Th,were decreased in the maxillary first molar region of the rats with periodontitis.Bone resorption in the root bifurcation area was significant and the distance from the CEJ to the ABC was significantly increased.After the GA intervention,all these indicators were reversed.（3）H&E and TRAP staining results.Compared with the control group,the epithelial integrity of the maxillary first molar was destroyed,the attachment loss was severe,the epithelial pegs in the gingival tissue were significantly extended and infiltrated with a large number of inflammatory cells in the lamina propria,and the number of activated osteoclasts on the alveolar bone surface was increased in the periodontitis group.However,the above pathological manifestations were improved after GA intervention.4.The regulatory role of the Cx43/JNK/NF-κB signaling pathway in the progression of periodontitis.Compared with the control group,the expression levels of Cx43,P-JNK,and NF-κB were significantly increased in the periodontal tissues of rats in the periodontitis group.All three expression levels decreased after pharmacological intervention with GA.5.Effect of Cx43/JNK/NF-κB signaling pathway on oxidative stress and apoptosis levels in periodontitis.Compared with the control group,the mitochondria in the periodontal tissue of rats in the periodontitis group exhibited significant swelling and vacuolization,mitochondrial membrane potential decreased,ROS content,expression levels of 8-OHd G,Bax,Caspase3,and TUNEL positive cells increased significantly,and the expression levels of SOD1 and anti-apoptotic protein Bcl-2 decreased;while all of the above indicators were reversed after GA intervention.Conclusions:1.Cx43 expression and function inhibition may effectively attenuate H₂O₂-induced oxidative damage in hPDLCs.2.The JNK/NF-κB signaling pathway may interact with Cx43 to co-regulate the redox status and apoptosis of hPDLCs.3.GA may effectively reduce periodontitis in rats.4.Inhibition of the Cx43/JNK/NF-κB signaling pathway may attenuate oxidative stress levels and apoptosis levels in periodontitis.5.The Cx43/JNK/NF-κB pathway plays a key role in promoting periodontitis progression while inhibiting this pathway can effectively alleviate periodontitis.