Fig 2: Inhibitory effects of geniposidic acid (GPA) on a Porphyromonas gingivalis-induced mouse periodontitis model. The mice were treated with P. gingivalis in the presence or absence of GPA and sacrificed, and the serum, gingival tissue, and upper molar bone were collected ((a) PBS inoculation; (b): GPA inoculation; (c): P. gingivalis inoculation; (d): GPA + P. gingivalis inoculation, n = 6 /group). Destruction of the mouse periodontal tissue was visualized in hematoxylin and eosin-stained sections (e): PBS inoculation, (f): GPA inoculation, (g): P. gingivalis inoculation, (h): GPA + P. gingivalis inoculation). Original magnification ×100; Scale bar = 100 µm. The inflammation score in sections from each group was evaluated (i). Data represents the mean ± SD of six mice per group. (i). Alveolar bone level of the upper jaw in each group (j). The amount of IL-6 in each group was determined using ELISA (k: gingival tissue, l: serum). In the boxplot, the horizontal black lines within each box denote the median values, and boxes are extended from the 25th and 75th percentile. Whiskers above and below the boxes indicate the 10th and 90th percentiles, respectively, and cross marks denote the averages (j–l). (m) Production of TLR2 in mouse gingival tissue of a representative sample (n = 3/group) with or without P. gingivalis inoculation in the presence or absence of GPA; the density of each band was measured by NIH imaging (n). Statistical analyses were performed using one-way ANOVA with Tukey–Kramer post hoc test (* p < 0.01, ** p < 0.05).

Fig 3: Transfection and silencing of TLR2 expression after siRNAs in BEL-7402.I, RT-PCR of TLR2 from pGenesil-1 vector, ScrambledsiRNA, sh-TLR2 RNAi(A, B, C) transfected BEL-7402. II, expression of TLR2 at mRNA level in pGenesil-1 vector, ScrambledsiRNA, and sh-TLR2 RNAi(A, B, C) transfected BEL-7402 with real-time PCR. III, flow cytometry analysis of TLR2 expression, student’s t-test and ANOVA were used to determine significance.

Fig 4: Inhibitory effects of geniposidic acid (GPA) on human gingival epithelial cells (HGECs) (a) mRNA expression of IL-6 in HGECs with or without Porphyromonas gingivalis stimulation (12 h) in the presence or absence of GPA (100 µg/mL). (b) Levels of IL-6 in the supernatant of HGECs with or without P. gingivalis stimulation (24 h) in the presence or absence of GPA (100 µg/mL). (c) Production of TLR2 in HGECs with or without P. gingivalis stimulation (12 h) in the presence or absence of GPA (100 µg/mL). (d–f) Levels of phosphorylated protein in HGECs with or without P. gingivalis stimulation (10 min) in the presence or absence of GPA (100 µg/mL, (d): p38, (e): JNK, f: ERK). Results are shown as the mean ± SD. * Significantly higher expression by Student’s t-test (* p < 0.01).

Fig 5: TLR2 expression and functional effect after TLR2 knockdown in BEL-7402 cell and in vivo.I, MTT analysis of the proliferative rate of pGenesil-1 vector, ScrambledsiRNA and sh-TLR2 RNAi(B) transfected cells. II and III, IL-6 and IL-8 presence in the supernatant secreted by pGenesil-1 vector, Scrambled siRNA and sh-TLR2 RNAi(B) transfected cells. Cell supernatant was collected and analyzed using flow cytometry. All results are representative of three separate experiments, student’s t-test and ANOVA were used to determine significance. IV, immunofluorescence analysis of gene-specific siRNA on TLR2 protein expression in pGenesil-1 vector(i), Scrambled siRNA(ii) and sh-TLR2 RNAi(B) (iii) transfected cells. Nuclear staining performed using DAPI (blue) (200×). TLR2 silencing suppresses tumor growth in an mouse model. Comparison of volume of tumors from each treatment group was shown. Treatment with BEL-7402 cell (VI_i), sh-TLR2 RNAi(B) constructive DNA was injected in tumor group(VI_ii), Significant differences from control groups(V.?, VI_iii) were represented by asterisks (P<0.05).