| Objective:The organization of hypoxia and cell autophagy is closely related to pulpitis,but the specific roles and regulatory mechanisms have not been fully elucidated.This study aims to evaluate the cellular damage,autophagy,and specific effects caused by hypoxia during the development of pulpitis based on clinical tissue specimens and in vivo and in vitro experiments.Additionally,it seeks to preliminarily explore the mechanisms by which the HIF-1α/BNIP3 signaling pathway participates in autophagy regulation.Methods:1.Based on clinical tissue specimens,the hypoxic state and autophagy level in pulpitis are investigated:(1)Collect pulp tissue specimens of the third molars from 40 patients aged 20-35,including 20 cases of pulpitis and 20 cases of normal controls.Five cases from each group are randomly selected for transcriptomics sequencing,and differential expression genes,related signaling pathways,and core gene proteins between the two groups are analyzed through GO,KEGG enrichment,and construction of a PPI network using bioinformatics;(2)In terms of morphological characteristics of tissue specimens,inflammation features are detected using H&E staining,and changes in ultrastructure are observed using electron transmission microscopy.Immunohistochemical staining techniques are simultaneously used to validate the expression of key pathways and core gene proteins enriched by transcriptomics sequencing in tissue specimens.2.Verification of hypoxia status and autophagy activation in pulpitis in animal models:(1)A pulpitis model in SD rats was established using the pulp cavity opening method,with 30 male SD rats aged 6-8 weeks randomly divided into a blank control group and pulp opening groups at 1,3,5,and 7 days,each group consisting of 6 rats.The blank control group received no treatment,while the model group rats underwent pulp opening on the left maxillary first molar according to the corresponding modeling time,with specimens collected and prepared accordingly;(2)H&E staining was used to assess the inflammatory progression in rat dental pulp tissues;(3)ELISA,MOD,and SDA assay kits were utilized to detect changes in inflammation and oxidative stress levels in rat serum;(4)Immunohistochemistry staining was performed to determine the expression levels of hypoxia and autophagy-related proteins HIF-1α,BNIP3,LC3B,and P62 in rat dental pulp tissues.3.Explore the damaging effects of hypoxia on cells and the specific role of autophagy at the cellular level:(1)Primary cells were isolated from human dental pulp tissue and identified;(2)The hypoxia model of human dental pulp cells was established to evaluate the changes of cell state:observe cell morphology changes under a light microscope,detect cell proliferation activity levels using the CCK-8 method,measure apoptosis levels using flow cytometry and JC-1 staining,mark cells with DCFH-DA to detect intracellular reactive oxygen species levels using flow cytometry and fluorescence staining,utilize ELISA to detect changes in intracellular inflammatory factors IL-6,IL-1β,TNF-α,and employ Western blot to assess changes in apoptosis and inflammation-related proteins Bax,Bcl-2,TNF-α,NF-κB expression levels;(3)To judge the changes of autophagy and autophagic flux induced by hypoxia:the structure of autophagy in cells was observed by MDC staining and electron transmission microscopy,the expression levels of LC3B and P62 were detected by immunofluorescence staining and Western blot.After 3-MA and CQ were used to intervene the cells,the activation of autophagic flux was judged by the changes of autophagy structure and related protein level(4)Investigate the specific role of autophagy in cell hypoxic conditions:cells were treated with autophagy inhibitors 3-MA and CQ under normoxic and hypoxic conditions.After inhibiting autophagy,changes in cell proliferation,apoptosis levels,intracellular reactive oxygen species,mitochondrial membrane potential,secretion of inflammatory factors,and expression levels of apoptotic and inflammatory-related proteins were detected through a series of molecular biology detection methods,followed by statistical analysis.4.Hypoxic intervention was applied on a rat model of dental pulpitis,combined with a cellular hypoxia model to study the regulatory mechanism of autophagy in dental pulpitis:(1)24 male SD rats aged 6-8 weeks were randomly divided into 4 groups,with 6 rats in each group.The blank control group was raised in a conventional animal room;the pulp exposure group had the first molar of the left maxilla opened and then raised in a conventional animal room;the hypoxia group of rats was raised in a low oxygen chamber(DYC-3013M)in the northwest region without any other interventions;the hypoxia+pulp exposure group had the first molar of the left maxilla opened and then raised in the low oxygen chamber.The intervention time was 7 days;(2)H&E staining was used to observe the inflammatory characteristics of rat dental pulp tissue,while immunohistochemical staining was used to detect the localization and expression levels of hypoxia and autophagy-related proteins HIF-1α,BNIP3,and LC3B in dental pulp tissue;(3)Based on the cellular hypoxia model,the HIF-1α chemical inhibitor YC-1 was used to intervene in cells,and immunofluorescence staining and Western blot were used to detect the expression levels of hypoxia and autophagy-related proteins.Results:1.The expression levels of hypoxia and autophagy are upregulated in human dental pulpitis tissues:(1)Transcriptomic sequencing identified 1351 differentially expressed genes,which are closely related to inflammation,immunity,autophagy,and mitophagy processes.Among them,the expression of BNIP3 protein,which is associated with both hypoxia and autophagy,is upregulated in the dental pulpitis group;(2)Compared to normal dental pulp tissues,the inflammatory dental pulp shows tissue structural damage,vascular dilatation with congestion in the lesion area,and extensive infiltration of inflammatory cells,with significant expression of typical autophagosome ultrastructure in the dental pulpitis tissues;(3)The expression levels of hypoxia-related protein HIF-1α,BNIP3,and autophagy-related proteins LC3B,P62,and BNIP3 are significantly upregulated in the dental pulpitis group compared to the normal group(P<0.05).2.In rats with pulpitis,hypoxia and autophagy activation were observed:(1)In the blank control group,normal tissue structure was visible in the pulp cavity,without inflammatory exudation or vascular dilation.However,with the progression of the modeling time,pulp tissue inflammation gradually spread from the crown to the root;(2)Serum testing results showed that,compared to the control group,the levels of inflammatory factors IL-6,IL-1β,and TNF-αwere significantly elevated(P<0.05).Meanwhile,the SOD activity in the serum of the model group significantly decreased,MDA levels increased,and the ratio of the two decreased(P<0.05);(3)In the tissues of rats in the pulpitis group,a large number of HIF-1α,LC3B,and BNIP3 positive cells were observed in odontoblasts,multi-layered cells,and fibroblasts,mainly expressed in the cytoplasm.In contrast,the expression of P62 protein in the blank control group was significantly higher,with statistically significant differences between groups(P<0.05).3.Hypoxia induces damage effects on human dental pulp cells and activates protective autophagy within the cells:(1)Primary human dental pulp cells were isolated and cultured in vitro,with a positive rate of CD29,CD90,and CD 105 surface antigens on mesenchymal cells exceeding 95%,displaying characteristics of odontoblastic,adipogenic,and chondrogenic differentiation potentials;(2)After 24 hours of hypoxic intervention,the cells exhibited disordered alignment,shrinkage,and other damage morphologies,accompanied by a decrease in proliferation activity.This also led to an increase in apoptosis rate,an upregulation of intracellular reactive oxygen species levels,showing statistical differences compared to the control group(P<0.05).Results from JC-1 staining revealed a transition from red fluorescence to green fluorescence in hypoxic group cells,indicating a decrease in mitochondrial membrane potential.Simultaneously,the expression levels of pro-inflammatory cytokines IL-6,IL-1β,TNFα showed an upward trend in the hypoxic group compared to the control group(P<0.05),accompanied by an increase in the expression levels of related proteins TNFα,NF-κB(P<0.05).Within the hypoxic group,the expression level of pro-apoptotic protein Bax was upregulated(P<0.05),while the expression level of anti-apoptotic protein Bcl-2 showed a decreasing trend(P<0.05);(3)Remarkable autophagic structures including autophagic acidic vesicles and autophagosomes were observed in the hypoxic group.Following hypoxic intervention,a large number of distinct green fluorescence-labeled LC3B protein puncta were induced,clustered and distributed in the cytoplasm.Western blot results further confirmed that compared to the control group,the expression level of autophagy-related protein LC3B-Ⅱ increased in the hypoxic group(P<0.05),along with a decrease in the level of P62(P<0.05).Upon pretreatment of cells with 3-MA,CQ,it was observed that the elevation of LC3B induced by hypoxia was due to the activation of intracellular autophagic flux,rather than inhibition of the lysosomal degradation pathway;(4)After inhibiting autophagy expression with 3-MA,CQ,the proliferation activity of cells continued to decrease,the apoptosis rate further increased,accompanied by an increase in intracellular reactive oxygen species release,leading to a further decline in mitochondrial membrane potential.Additionally,the levels of inflammatory factors within the cells and the expression of inflammatory,apoptosis-related proteins showed a significant further upward trend compared to the control and hypoxic groups(P<0.05).4.Hypoxia-induced autophagy in pulpitis is mediated by the HIF-1α/BNIP3 signaling pathway:(1)H&E staining results showed that in the hypoxia group of rat dental pulp,there were morphological changes,with a large number of inflammatory cells infiltrating the crown and one-third of the root,accompanied by vascular dilation and blood clot formation.In the hypoxia+pulp exposure group,necrosis was observed in the dental pulp tissue below the exposed point of the crown,with a large amount of inflammatory cells clustering at the root canal orifice,and a significant number of red blood cells spreading to the lower part of the root canal.In addition,immunohistochemical staining revealed that HIF-1α,BNIP3,and LC3B positive cells were significantly expressed in odontoblasts and fibroblasts of the hypoxia and hypoxia+pulp exposure groups,mainly concentrated in the cytoplasm and cell membrane,with statistical differences between the blank control and pulp exposure groups(P<0.05);(2)After inhibiting HIF-1α expression with YC-1,immunofluorescence and Western blot confirmed that downregulation of HIF-1α directly led to a synchronous decrease in BNIP3 protein and autophagy-related protein LC3B-Ⅱ levels.Conclusion:Hypoxia and autophagy are involved in the occurrence and development of pulpitis,with hypoxia-induced autophagy being protective.The HIF-1α/BNIP3 pathway is involved in regulating autophagy in pulpitis.This provides a new molecular explanation for the activation of autophagy in pulpitis and offers new insights for understanding the pathogenesis of pulpitis and developing protective treatments for dental pulp. |
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