Effects of Porphyromonas gingivalis injected through tail vein on the expressions of biomarkers in neural stem cells and neurons of wild-type rats hippocampus / 中华口腔医学杂志

Objectives: To study the effects of Porphyromonas gingivalis (Pg) injected through tail vein on the molecular expression levels of biomarkers of neural stem cells (NSC) and neurons in the hippocampus of wild-type adult rats, and the effects on hippocampal neurogenesis. Methods: Eighteen male Sprague-Dawley (SD) rats were randomly divided into 3 groups based on the table of random numbers (n=6 in each group). In low-intensity group and high-intensity group, rats were injected intravenously through tail vein with 200 μl Pg ATCC33277 [1.0×103 and 1.0×108 colony forming unit (CFU), respectively] 3 times per week for 8 weeks. In the sham group, 200 μl of phosphate buffer saline (PBS) was given instead. Behavioral tests: the navigation and the exploration tests using Morris water maze (MWM) were applied to evaluate learning and memory ability of rats. Immunohistochemistry was performed to detect cells positively expressing nestin, doublecortin (DCX) and neuronal nuclei (NeuN) in the subgranular zone (SGZ) of rats in each group. Western blotting was used to evaluate the expression levels of nestin, DCX and NeuN in rat hippocampus. Results: Learning and memory abilities: on day 5 of navigation test, the lagency time was 22.83 (16.00, 38.34) s in the high-intensity group, significantly longer than the sham group [5.59 (5.41, 6.17) s] (t=-11.17, P<0.001). There were no significant differences between the low-intensity group [9.85 (8.75, 21.01) s] and the sham group (t=-6.83, P=0.080). Results in the exploration test showed that, in the high-intensity group, the number of fime crossing over the previous platform area within 60 s was 1.50 (1.00, 2.00), significantly less than the sham group [4.00 (2.75, 4.00)] (t=9.75, P=0.003); no significant differences between the low-intensity group [2.50 (2.00, 3.00)] and the sham one (t=4.50, P=0.382). Immunohistochemistry showed that the nestin+ cell density in the low-intensity group [(35.36±4.32) cell/mm2] and high-intensity group [(26.51±5.89) cell/mm2] were significantly lower than the sham group [(59.58±14.15) cell/mm2] (t=24.21, P=0.018; t=33.07, P=0.005); as for the mean absorbance of DCX+ cells, the low-intensity group (0.007±0.002) and the high-intensity group (0.006±0.002) were significantly lower than the sham group (0.011±0.001) (t=0.004, P=0.018; t=0.006, P=0.005); compared with the sham group [(1.13±0.14)×103 cell/mm2], the density of NeuN+ neurons in the high-intensity group [(0.75±0.08)×103 cell/mm2] was significantly reduced (t=0.38, P=0.017), and was not significantly changed in the low-intensity group [(0.88±0.19)×103 cell/mm2] (t=0.25, P=0.075). Western blotting results showed that, compared with the sham group, the expression levels of nestin, DCX, and NeuN were significantly reduced in the high-intensity group (t=0.74, P<0.001; t=0.18, P=0.014; t=0.35, P=0.008), but were not statistically changed in the low-intensity group (t=0.18, P=0.108; t=0.08, P=0.172; t=0.19, P=0.077). Conclusions: Pg injected through tail vein may reduce learning and memory abilities of wild-type rats, and may reduce the number of nestin, DCX, and NeuN-positive cells, and the protein expression levels of the above molecules in the hippocampus.

17-β estradiol promotes the expression of interleukin-6 in human periodontal ligament cells infected with Porphyromonas gingivalis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of 17-β estradiol (E(2)) and Porphyromonas gingivalis (Pg) W83 on the expression of interleukin (IL)-6 and IL-8 in human periodontal ligament cells (hPDLC).</p><p><b>METHODS</b>Primary cultures of hPDLC were established and the cells of passage four were treated with 10(-10) mol/L E(2), 10(-7) mol/L E(2) or PgW83 individually or E(2) combined with PgW83. The expression levels of IL-6 and IL-8 protein at 12 h and 24 h were measured with enzyme-linked immunosorbent assay and the levels of mRNA at 24 h were detected with real-time reverse transcriptase polymerase chain reaction.</p><p><b>RESULTS</b>The expression level of IL-6 reached (2482.88 ± 26.53) ng/L in hPDLC treated with Pg at multiplicity of infection (MOI) of 100 for 24 h, which was significantly higher than that in hPDLC treated with Pg at MOI of 10:1 [(734.09 ± 87.90) ng/L, P = 0.000], the controls [(425.8 ± 77.25) ng/L, P = 0.000] and that in hPDLC treated with Pg at MOI of 100 for 12 h [(1157.50 ± 234.65) ng/L, P = 0.000]. The expression level of IL-8 reached (4965.81 ± 1072.55) ng/L in hPDLC treated with Pg at MOI of 100 for 24 h, which was significantly higher than that in hPDLC treated with Pg at MOI of 10 [(803.51 ± 162.08) ng/L, P = 0.007], the controls [(400.75 ± 2.27) ng/L, P = 0.005] and that in hPDLC treated with Pg at MOI of 100 for 12 h [(1431.12 ± 82.78) ng/L, P = 0.001]. E(2) did not show remarkable effect on the expressions of IL-6 and IL-8. E(2) combined with Pg (MOI = 100:1) significantly promoted the expression levels of IL-6 at 24 h while did not influence those of IL-8. The relative mRNA level of IL-6 in hPDLC treated with 10(-10) mol/L E(2) or 10(-7) mol/L E(2) combined with Pg were 0.49 ± 0.15 (P = 0.021)and 0.53 ± 0.16 (P = 0.036) individually, which were significantly higher than that treated with Pg alone, 0.19 ± 0.06. The protein level of IL-6 in hPDLC treated with 10(-10) mol/L E(2) or 10(-7) mol/L E(2) combined with Pg were (5512.66 ± 1022.07) ng/L (P = 0.012) and (6988.78 ± 2279.13) ng/L (P = 0.000) individually, which were significantly higher than that treated with Pg alone, (3138.46 ± 183.72) ng/L.</p><p><b>CONCLUSIONS</b>PgW83 significantly increased the expression levels of IL-6 and IL-8 in hPDLC in a dose-and time-dependent manner. Without the infection of periodontal pathogens, estrogen may exert no effect on the expression of IL-6 and IL-8 while it may promote the expression of IL-6 in hPDLC when combined with Pg, which may in turn promote the process of periodontal inflammation.</p>

Effects of calcitriol on the expression of vitamin D receptor, RANKL and osteoprotegerin in human periodontal ligament cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the effects of 1, 25-dihydroxyvitamin D(3) (VD(3)) on the expression of vitamin D receptor (VDR), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament cells (hPDLC) populations and to analyze the potential mechanisms.</p><p><b>METHODS</b>Twelve hPDLC populations were primarily established from 12 donors individually. Two samples of each hPDLC population of passage three were treated respectively with 10(-8) mol/L VD(3) (V D(3) group) or 0.1% absolute ethyl alcohol as controls (control group). Six days later, the mRNA expression levels of VDR, RANKL and OPG in the samples were determined with real-time quantitative RT-PCR. The DNA base sequences upstream to the transcription start site of RANKL gene were also analyzed.</p><p><b>RESULTS</b>Compared with the control group, the mRNA expression level of VDR increased significantly in the VD(3) group (P = 0.003), averagely (3.04 +/- 1.06) times of that in the control group; the mRNA expression level of RANKL was also up-regulated by VD(3) (P = 0.001), 9.82 (0.75-119.18) times of that in the control group; the OPG expression level was (94.48 +/- 39.15)% of the controls (P = 0.136); OPG/RANKL ratio was down-regulated in the VD(3) group to averagely 10.36% (1.01%-138.00%) of the controls (P = 0.003). No mutation was found in the DNA fragments upstream to the transcription start site in the RANKL gene and the genotypes of the polymorphism at -1832 (rs7984870, C/G) were not shown to be significantly related to the RANKL mRNA expression level.</p><p><b>CONCLUSIONS</b>In hPDLC, VD(3) can significantly increase the mRNA expression level of VDR; VD(3) can increase RANKL mRNA expression level to decrease OPG/RANKL ratio, but it has little effect on OPG mRNA expression. The big differences of the RANKL mRNA regulation in response to VD(3) treatment among hPDLC populations may not be associated with the DNA sequences upstream to the transcription start site in the RANKL gene.</p>