[Effect of galectin-3 on lipopolysaccharide-induced proliferation, migration, apoptosis, reactive oxygen species and inflammatory cytokine production in human gingival fibroblasts].

Abstract

Objective: To investigate the effects of galectin-3 (Gal-3) expression on lipopolysaccharide (LPS)-induced proliferation, migration, apoptosis, reactive oxygen species (ROS) and inflammatory cytokine production in human gingival fibroblasts (GF) as well as its mechanism, thus laying the foundation for an in-depth discussion of the regulatory role of Gal-3 in periodontitis and its mechanisms. Methods: Gingival tissues from 6 periodontally healthy subjects undergoing crown lengthening were collected at the Department of Periodontology, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University from December 2022 to December 2023. GFs were extracted and cultured by collagenase digestion. Lentivirals with multiplicity of infection (MOI) of 15, 20, 30, 40, 50, 60, 70, 80 were used to achieve knockdown and overexpression of Gal-3 gene in GFs, whose efficiencies of Gal-3 gene were detected by using immunofluorescence, real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Negative control of knockdown (shNC)+LPS group, Gal-3 knockdown (shGal-3)+LPS group, negative control of overexpression (oeNC)+LPS group, and Gal-3 overexpression (oeGal-3)+LPS group were established, respectively. 5-Ethynyl-2'-deoxyuridine (EdU), Ki67 staining, scratch migration assay, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technology, immunofluorescence assay and RT-qPCR were used to investigate the effects of Gal-3 on LPS-induced proliferation, migration, apoptosis, ROS, interleukin (IL)-6, IL-8 expression. The effects of Gal-3 knockdown on the expression of differential genes and the enrichment of signaling pathways in LPS-induced GFs were investigated by RNA sequencing (RNA-seq). Results: More than 80% of GFs were successfully transfected by shGal-3 MOI 40 and oeGal-3 MOI 70. Immunofluorescence results showed that the morphologies of GFs were normal after lentiviral transfection, and green fluorescence could be distributed in the cytoplasm, nucleus, and cell membrane. The results of RT-qPCR and Western blotting assay showed that the expressions of Gal-3 at the gene and protein levels in shGal-3 group (0.26±0.01, 0.26±0.03, respectively) were significantly lower than those in the shNC group (1.00±0.03, 1.00±0.09, respectively) (P<0.001); the expressions of Gal-3 at the gene and protein levels in the oeGal-3 group (4.26±0.05, 3.94±0.34) were significantly higher than those in the oeNC group (1.00±0.00, 1.00±0.24, respectively) (P<0.001). EdU, Ki67 experiments showed that the percentage of GFs proliferation was significantly lower in the shGal-3+LPS group [(16.99±1.79)%, (13.48±0.95)%, respectively] than in the shNC+LPS group [(33.86±3.84)%, (35.63±1.62)%, respectively] (P<0.05), and the proliferation ratio of GFs was significantly increased in the oeGal-3+LPS group [(45.36±1.56)%, (45.83±1.50)%, respectively] compared to the oeNC+LPS group [(34.47±1.02)%, (33.66±3.14)%, respectively] (P<0.05). The results of scratch migration assay showed that the migration ratio of GFs in shGal-3+LPS group significantly decreased compared to the shNC+LPS group [(25.07±0.01)% vs (57.84±0.00)%] (P<0.001), whereas the soeGal-3+LPS group significantly facilitated the migration ratio of GFs compared to the oeNC+LPS group [(74.70±0.03)% vs (53.36±0.01)%] (P<0.001). The results of TUNEL experiments showed that LPS stimulation with shGal-3 promoted apoptosis of GFs (P<0.05), whereas oeGal-3 inhibited apoptosis of GFs (P<0.001). Immunofluorescence experiments and RT-qPCR results showed that knockdown of Gal-3 significantly reduced ROS production, IL-6 and IL-8 expression levels at the gene level in GFs (P<0.001), whereas overexpression of Gal-3 significantly increased the production of ROS and the expression of IL-6 and IL-8 at the gene level in GFs (P<0.001). RNA-seq results showed that differential genes caused by Gal-3 knockdown under LPS conditions were significantly enriched in biological processes such as cellular response to type Ⅰinterferon in the Gene Ontology database and in the Kyoto Encyclopedia of Genes and Genomes database for NOD-like receptor, RIG-I like receptor and other signaling pathways. Conclusions: Gal-3 knockdown inhibited LPS-induced proliferation, migration, ROS, IL-6 and IL-8 production, and promoted apoptosis of GFs, while overexpression had the opposite effect. This process might be closely linked to the Janus kinase-signal transducer and activator of transcription pathway.