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JKM > Volume 39(1); 2018 > Article
Park, Lee, and Yang: The effects of Eclipta Prostrata L.(Ecliptae Herba) on periodontitis rats

Abstract

Objectives

Periodontitis is an inflammatory disease with the destruction of periodontal ligament, alveolar bone loss and inflammation of gingva, leading to teeth loss. Eclipta prostrata L. (Ecliptae Herba) has been used to treat the inflammatory disease as a Korean traditional medicine. The aim of this study is to investigate the effects of E. prostrata L. on periodontitis.

Methods

E. prostrata L. was extracted with water and lyophilized. The aqueous extract of E. prostrata L. (EP) was topically applied to the periodontal lesion for 2 weeks. To induce the periodontitis, a 3-0 nylon ligature was placed around the cervix of the lower first molar in rat. Rats were divided into 3 groups (n = 7); NL group (non-ligatured and non-treated), L group (ligatured and vehicle-treated) and EP group (ligatured and EP-treated). After sacrifice, the mandibles was dissected and stained with methylene blue solution to analyze the alveolar bone loss. The expression of MMP-9 was determined in gingival tissues. To confirm the effect of EP on recovery of gingiva, mRNA expressions of type I pro-collagen and MMP-9 levels were investigated in LPS-treated HS68 fibroblast cells. In addition, inflammatory mediators were evaluated in LPS-treated RAW264.7 cells.

Results

Alveolar bone loss was significantly inhibited by EP treatment. The mRNA expression of MMP-9 was attenuated in rats treated with EP. In addition, treatment with EP increased the expression of type I pro-collagen, while the expression of MMP-9 was decreased in LPS-stimulated HS68 fibroblast cells. Furthermore, EP down-regulated the LPS-induced IL-6, TNF-α, COX-2 and iNOS production in RAW264.7 cells.

Conclusions

These results suggest that EP have ameliorative effects on periodontitis through inhibiting alveolar bone loss and modulating the inflammatory mediators. Therefore, E. prostrata L. may be an alternative on patients with periodontitis.

Fig. 1
Experimental design for induction of periodontitis.
Ligature was placed around the cervix of the bilateral mandibular first molars of rats.
jkm-39-1-63f1.gif
Fig. 2
Effects of EP on alveolar bone loss. (A) Representative images and (B) quantified values of alveolar bone loss. The red lines represent the distance from the cementoenamel junction to the alveolar bone crest. ###p < 0.001 vs. NL; **p < 0.01 vs. L. NL, non-ligatured and non-treated; L, ligatured and vehicle-treated; EP, ligatured and Eclipta prostrata-treated.
jkm-39-1-63f2.gif
Fig. 3
Effects of EP on the MMP-9 expression in gingival tissues. GAPDH has been used as loading control. ###p < 0.001 vs. NL; ***p < 0.001 vs. L. NL, non-ligatured and non-treated; L, ligatured and vehicle-treated; EP, ligatured and Eclipta prostrata-treated.
jkm-39-1-63f3.gif
Fig. 4
Effects of EP on the expressions of type I pro-collagen and MMP-9 in LPS-stimulated HS68 fibroblast cells.
(A) Type I pro-collagen and (B) MMP-9 expressions. GAPDH has been used as loading control. ###p < 0.001 vs. normal; ***p < 0.001 or *p < 0.05 vs. LPS. EP; Eclipta prostrata L. extract.
jkm-39-1-63f4.gif
Fig. 5
Effects of EP on the expressions of IL-6 and TNF-α in LPS-stimulated RAW264.7 macrophage cells. (A) IL-6 and (B) TNF-α expressions. ###p < 0.001 vs. normal; ***p < 0.001 vs. LPS. EP; Eclipta prostrata L. extract.
jkm-39-1-63f5.gif
Fig. 6
Effects of EP on the expressions of iNOS and COX-2 in LPS-stimulated RAW264.7 macrophage cells. (A) iNOS and (B) COX-2 expressions. β-actin has been used as loading control. ###p < 0.001 vs. normal; ***p < 0.001 vs. LPS; *p < 0.05 vs. LPS. EP; Eclipta prostrata L. extract.
jkm-39-1-63f6.gif
Table 1
RT-PCR Sequence of Target Designed by the Software of Applied Bioscience Prism 7000 System.
Target gene Amino acid sequences Amplicon size (bp)
MMP-9 (forward) GGG ACG CAG ACA TCG TCA TC
(reverse) TCG TCA TCG TCG AAA TGG GC
300–500
Type I pro-collagen (forward) TCT ACT GGC GAA ACC TGT ATC CG
(reverse) CAA GGA AGG GCA GGC GTG AT
2000
GAPDH (forward) CCA TCA TCT TCC AGG AG
(reverse) CCT GCT TCA CCA CCT TCT TG
576

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