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Experimental Study of Protective Effects of Baicalin on Human Periondontal Ligament Fibroblasts

Author: LiHuiYing
Tutor: ZhaoManLin；ChenHuiZhen
School: Hebei Medical University
Course: Clinical Stomatology
Keywords: baicalin lipopolysaccharide human periodontal ligament fibroblasts tumor necrosis factor alpha induce
CLC: R285.5
Type: Master's thesis
Year: 2005
Downloads: 105
Quote: 3
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Abstract

Obiective: Periodontitis is an inflammatory disease that leads to irreversible periodontal pockets, attachment loss, bone destruction and eventually tooth loss, which is one of the most significant causes of tooth loss in adults. Studies have showed that bacteria gathering in the plaque are implicated in the development of periodontal disease, especially the gram-negative anaerobic bacteria. Endotoxin is a major cell wall component of gram-negative bacteria, which is called lipopolysaccharide (LPS). The poison action of LPS has directly effects on development of human periodontal ligament fibroblasts (HPLFs) and stimulates macrophages, monocytes and fibroblasts to secrete a variety of proinflammatory cytokines, such as interleukin-1 (IL-1), tumor necrosis factor (TNF-α) and prostaglandin E2 (PGE2) , so there is immediate significance to resist destruction of LPS to HPLFs by antagonism of drug. Scutellaria baicalensis is a traditional Chinese medicine and one of its major components is baicalin that has a variety of physiological functions such as antibacterial and anti-inflammatory functions. By the present experiment we observed the effect of baicalin on LPS which decreases proliferation activities of HPLFs and leads to the impairments of HPLFs, and the effect of baicalin on HPLFs activities secreting TNF-αinduced by LPS of Escherichia coli (E.coli) in vitro, examined protecting action of baicalin to HPLFs, preliminarily explored possible pathogenesis of periodontitis and provided experimental and theorial basis of a new drug for preventing and treating periodontal disease. Methods: Connective tissue cells for this study were obtained from healthy periodontal ligament of pre-molar teeth of individuals undergoing tooth extractions for orthodontic reasons, the periodontal ligament tissue attached to the middle third of the root was removed by scraping with a sterilized scalpel, HPLFs were cultured in cell culture technique and examined by immunohistochemical staining with vimentin and keratin antibody, morphological characteristics of HPLFs were observed in light microscope by HE staining. Enzyme-linked immunosorbent assay (ELISA), MTT method and transmission electron microscope (TEM) method were used to observe the effects of baicalin on LPS.There were five treatment groups in this study: control(group A); group treated with 100μg/ml LPS(group B); groups treated with different concentration baicalin (group C); groups treated with 100μg/ml LPS and different concentration baicalin (group D); group treated with 100μg/ml LPS and 2μg/ml dexamethasone (group E). Baicalin final concentrations are 10, 1, 0.1, 0.01, 0.001μg/ml respectively. 1 HPLFs proliferation activity measurementThe fifth generation of HPLFs were obtained and seperated by 2.5 per cent trypsin at concentration of 3×104/ml, then they were incubated in 96-well culture plate, the volume of culture fluid was 100μl per well. After cells were incubated for 24 hours, culture supernatant was abandoned. Solution of each treatment group was added in 6 wells. After cells were incubated for 3 days, 20 μl MTT solution (5mg/ml) was added in per well and cells were incubated for 4 hours continuatively, then the supernatant in per well was abandoned, 150μl DMSO was added in per well. After the plate was vibrated for 10 minutes, HPLFs proliferation activity was determined in 96-well plate by measuring of absorbance at 490 nm and the mean of six wells was analyzed statistically. 2 TNF-αassay and dermination The fifth generation of HPLFs were obtained and seperated by 2.5 per cent trypsin at concentration of 4×104/ml, then they were incubated in 48-well culture plate, the volume of culture fluid was 0.5ml per well. After cells were incubated for 24 hours, culture supernatant was abandoned. Solution of each treatment group was added in 5 wells. After cells were incubated for 2 days, the culture supernatant was harvested and stored in -20℃refrigerator for use. TNF-αrelease by HPLFs was estimated with enzyme-linked immunosorbent assay according to the manufacturer’s instructions. 3 The effect of baicalin on ultrastruture of HPLFs induced by LPSThe fifth generation of HPLFs were obtained and seperated by 2.5 per cent trypsin at concentration of 1×104/ml, they were incubated in three 80ml culture flasks, then 4ml solution of treatment group A, treatment group B and treatment group D with 10 μg/ml baicalin was added in three flasks respectively. After incubated for 6 days, they were separated by 2.5 per cent trypsin and centrifuged, then were fixed, made into TEM specimen, observed and photoed in TEM. Results: After primary HPLFs were cultured successfully, the majority of HPLFs had a spindle-shaped, elongated appearance characteristic of fibroblast-like cells in light microscope, the minority were a star-shaped. By HE staining their plasma was pink and blue or purple nucleus was round or oval at the center of cells. Expression of vimentin was found and expression of keratin was not found in the plasma of HPLFs, which showed that HPLFs stemmed from mesoclerm, the fifth generation of HPLFs were used in this study. At the concentration of 0.001~10μg/mL, baicalin could improve proliferation activities of HPLFs significantly in comparison with controls (P<0.05). At the concentration of 100μg/mL, LPS of E.coli could inhibit the proliferation activities of HPLFs remarkably and proliferation activities of HPLFs improved greatly after adding baicalin and dexamethasone, baicalin and dexamethasone could significantly inhibit the activity of TNF-αinduced by LPS and significantly suppressed TNF-αproduction of HPLFs stimulated with LPS (P<0.01), but among the

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