The Effect Of Osmanthus Fragrans Ethanol Extract On Porphyromonas Gingivalis Lipopolysaccharide-stimulated Periodontal Inflammation

Periodontal disease is the primary cause of adult tooth loss. Porphyromonas gingivalis (P. gingivalis), gram negative bacteriais, the main pathogen of periodontal disease, has a high detection rate in the periodontal lesions. This bacteria can produce lipopolysaccharide, collagenase, gingipain, fimbriae and a series of virulence factors, induce many types of inflammatory factors, and damage periodontal support tissue. Not only so, in the periodontal inflammation, a large amount of reactive oxygen species can be generated, which will damage the balance of oxidation and antioxidation in the body. How to effectively remove excess free radicals and reduce the damage of periodontal tissues has gradually become a new focus in the prevention and treatment of periodontal inflammation.At present, searching for the natural drugs has attracted everyone’s attention. Previous studies have demonstrated that green tea, coffee, grape and other natural polyphenols can inhibit the growth of P. gingivalis and reduce the secretion of inflammatory factors. Osmanthus fragrans is a natural herbal medicine. It also has a certain effect on stomatitis and periodontitis. Xianning Hubei is the famous "township of the osmanthus fragrans "of China. The aim of this study was to prepare osmanthus ethanol extract using regional advantages and combining with local characteristics, to explore its cytotoxicity and effects on the proliferation and collagenase activity of P. gingivalis. It also tested the effects of osmanthus ethanol extract pretreatment on the expression of inflammatory cytokines, oxidative stress marker and cell protection enzyme induced by P. gingivalis lipopolysaccharide.The first chapter:the preparation of osmanthus ethanol extract and its toxic effect on human periodontal ligament cellsOsmanthus ethanol extract was prepared and human periodontal ligament cells were cultured in vitro. After the human periodontal ligament cells were cultured with the osmanthus ethanol extract, the cytotoxic effects of the osmanthus ethanol extract with different concentrations on the human periodontal ligament cells were investigated and analyzed by trypan blue staining method and Roche cell proliferation kit I (MTT). The results of the trypan blue staining showed that after co-cultured with different concentrations of the osmanthus ethanol extracts for 72 hours, the cell viability of human periodontal ligament cells was more than 95%. The analysis results of cell proliferation assay (MTT) showed that osmanthus ethanol extract did not have obvious cytotoxicity on human periodontal ligament cells. After co-cultured with the osmanthus ethanol extracts for 72 hours, in the concentration of 31.25μg/mL group, the relative proliferation rate of human periodontal ligament cells was 99.27±2.04%. In the concentration of 62.5μg/mL group, the cell relative proliferation rate was 98.26±1.69%. In the concentration of 125μg/mL group, the cell relative proliferation rate was 97.66±2.13%. In the concentration of 250μg/mL group, the cell relative proliferation rate was 97.04±2.43%. There was no significant difference between the experiment groups and the control group (P>0.05).After co-cultured with the osmanthus ethanol extracts and lipopolysaccharide for 72 hours, the cell viability was more than 75%. The experimental results showed that after co-cultured with the osmanthus ethanol extracts and lipopolysaccharide for 72 hours, in the concentration of 31.25μg/mL group, the relative proliferation rate of human periodontal ligament cells was 75.07±3.17%. In the concentration of 62.5 μg/mL group, the cell relative proliferation rate was 82.18±2.01%. In the concentration of 125μg/mL group, the cell relative proliferation rate was 93.54±2.70%. In the concentration of 250μg/mL group, the cell relative proliferation rate was 94.07±2.63% .62.5,125 and 250μg/mL osmanthus ethanol extract could significantly reduce the cytotoxicity induced by P. gingivalis lipopolysaccharide (P<0.05).125μg/mL osmanthus ethanol extract could completely reduce the cytotoxicity induced by P. gingivalis lipopolysaccharide (P<0.05).The results of this experiment were as follows:● The ethanol extract of osmanthus fragrans was prepared successfully● Co-cultured with different concentrations of the osmanthus ethanol extracts for 72 hours, the relative proliferation rate of human periodontal ligament cells was more than 97%.● The osmanthus ethanol extract could reduce the toxic effects of P. gingivalis lipopolysaccharide on human periodontal ligament cells.The second chapter:Effect of osmanthus ethanol extract on the growth and collagenase activity of P. gingivalisP. gingivalis was cultured in vitro. After P. gingivalis was co-cultured with different concentrations of the osmanthus ethanol extract, the effects of the extract on the growth and collagenase activity of P. gingivalis were investigated.The results showed that the ethanol extract of osmanthus significantly inhibited the growth of the bacteria (P<0.05). In the experimental results, under the THB-HK medium environment, the optical density value of P. gingivalis was 0.82±0.005. Co-cultured with 31.25μg/mL osmanthus ethanol extract, the optical density value was 0.77±0.025 and the inhibition rate was 5.47±3.11%. Co-cultured with 62.5 μg/mL osmanthus ethanol extract, the optical density value was 0.59±0.017 and the inhibition rate was 27.17±2.10%. Co-cultured with 125μg/mL osmanthus ethanol extract, the optical density value was 0.48±0.047 and the inhibition rate was 41.42±5.76%. Co-cultured with 250μg/mL osmanthus ethanol extract, the optical density value was 0.20±0.007 and the inhibition rate was 75.00±0.80%. It suggested that when the concentration of the ethanol extract was more than 62.5μg/mL, it can significantly inhibit the growth of P. gingivalis (P<0.05). In MBB-H medium, the optical density of P. gingivalis was 0.67±0.016. Co-cultured with 31.25μg/mL osmanthus ethanol extract, the optical density value was 0.45±0.030 and the inhibition rate was 32.79±4.48%. Co-cultured with 62.5 μg/mL osmanthus ethanol extract, the optical density value was 0.37±0.046 and the inhibition rate was 45.02±6.82%. Co-cultured with 125 μg/mL osmanthus ethanol extract, the optical density value was 0.18±0.032 and the inhibition rate was 72.86±4.79%. Co-cultured with 250 μg/mL osmanthus ethanol extract, the optical density value was 0.10±0.006 and the inhibition rate was 85.24±0.90%. It suggested that when the concentration of the ethanol extract was more than 31.25 μg/mL, it can significantly inhibit the growth of P. gingivalis (P<0.05). Therefore, the kind of bacteria medium could affect the inhibition of osmanthus ethanol extract on P. gingivalis.The results of collagenase activity testing showed that the ethanol extract of osmanthus significantly inhibited the activity of P. gingivalis collagenase. In the experimental results, the fluorescence values of the blank control group from the beginning to the end of 5h were as follows:1115±7.81,1147±7.37,1151±5.57, 1153±6.24,1156±6.81,1158±6.00,1162±6.03,1165±7.09,1169±5.51,1173±5.57, 1178±4.58; Bacterial control group:1173±5.57,1314±150.32,1494±16.37, 1555±19.52,1595±23.59,1625±24.27,1646±24.95,1665±25.58,1678±24.44, 1686±25.24,1700±26.51; Inhibitor group:1165±1.55,1227±21.28,1234±22.19, 1243±21.66,1254±21.07,1257±20.52,1258±21.28,1259±21.94,1262±21.73, 1266±22.14,1270±22.61; 250μg/mL osmanthus ethanol extract group:1148±2.52, 1220±3.51,1232±5.13,1246±5.69,1261±4.58,1268±7.55,1272±6.43,1277±7.09, 1281±8.00,1285±8.62,1289±9.54; 125μg/mL osmanthus ethanol extract group: 1194±2.65,1293±1.73,1311±2.08,1334±2.08,1353±2.31,1365±2.00,1373±3.61, 1379±3.51,1385±4.04,1393±5.69,1397±5.03; 62.5μg/mL osmanthus ethanol extract group:1152±25.54,1287±22.01,1326±23.90,1351±28.51,1376±27.00, 1389±26.51,1406±26.16,1417±26.63,1428±27.47,1436±26.85,1443±26.51; 31.25 μg/mL osmanthus ethanol extract group:1170±11.02,1367±26.31,1412±27.73, 1451±30.04,1478±28.88,1494±27.15,1508±27.73,1518±26.00,1530±26.00, 1538±26.00,1544±26.58. It suggested that the fluorescence value of each group increased rapidly in the early stage, and reached at balance after 4 hours. For the same time point, the higher the concentration of ethanol extract of osmanthus, the lower the fluorescence value. We selected the time point of the 5th hour to calculate the effect of different concentrations of ethanol extract of osmanthus on the activity of collagenase. The results showed that when the concentration of osmanthus ethanol extract was 31.25μg/mL, the collagenase activity rate was 70.11±5.09%. When the concentration was 62.5 μg/mL, the collagenase activity rate was 50.77±5.08%. When the concentration of 125 μg/mL, the collagenase activity rate was 41.95±0.96%. When the concentration was 250 μg/mL, the collagenase activity rate was 21.26±1.83%. It suggested that 31.25 μg/mL extract can inhibit 30% of P. gingivalis collagenase activity and 62.5μg/mL extract can inhibit 50% of P. gingivalis collagenase activity, 125μg/mL extract can inhibit 60% of P. gingivalis collagenase activity,250μg/mL extract can inhibit 80% of P. gingivalis collagenase activity. The osmanthus ethanol extract could significantly inhibit P. gingivalis collagenase activity (P<0.05).The results of this experiment were as follows:● P. gingivalis was cultured in vitro;● In THB-HK (iron rich) culture medium, the osmanthus ethanol extract was co-cultured with P. gingivalis for 48 hours. When the extract concentration was more than or equal to 62.5μg/mL, it could significantly inhibit bacterial growth. The inhibition rate was about 27% to 76%;● In MBB-H (iron deficiency) culture medium, the osmanthus ethanol extract was co-cultured with P. gingivalis for 48 hours. When the extract concentration was more than or equal to 31.25 μg/mL, it could significantly inhibit bacterial growth. The inhibition rate was about 33% to 85%;● The osmanthus ethanol extract could significantly inhibit P. gingivalis collagenase activity. The inhibition rate was about 30% to 80%.The third chapter:Effect of osmanthus ethanol extract on the secretion of inflammatory cytokines induced by P. gingivalis lipopolysaccharideThis part of the experiment was to explore the effects of different concentration of osmanthus ethanol extract on the secretion of inflammatory cytokines IL-6, IL-8 induced by P. gingivalis lipopolysaccharide, mainly using the human periodontal ligament cells in vitro as the research object, P. gingivalis lipopolysaccharide as inducing factor.The ELISA method was used in this experiment, and the concentration of IL-6 in the cell culture supernatant was detected by means of the commercial ELISA kit. The results showed that the concentration of IL-6 in the cell control group was 101.57±3.95 pg/mL, and the concentration of IL-6 in the LPS control group was 11444.93±76.36 pg/mL. The concentrations of IL-6 in 4 drug control groups (concentration from high to low) were 23.17±0.97,42.13±7.33,61.20±9.20, 102.47± 16.62 pg/mL。 The concentrations of IL-6 in the 4 experimental groups were 251.43±6.01,5373.17±180.95,11267.03±98.93,11525.37±82.49 pg/mL. Thus it could be seen that comparing to the cell control group, IL-6 concentrations in LPS control group were significantly higher (P<.05), indicating that P. gingivalis lipopolysaccharide could successfully induce human periodontal ligament cells to secrete IL-6, mediating periodontal inflammation. Compared with the cell control group, IL-6 concentrations in four drug control group were very low (three concentration, P<0.05), indicating that pure co-cultured with osmanthus ethanol extract did not have stimulation effect on IL-6 secretion of human periodontal ligament cells. Compared with the LPS group, only at the concentration of 125,250 μg/mL, IL-6 expression level decreased significantly, indicating that when the extract concentration is equal to or more than 125 μg/mL, the pretreatment can inhibit the expression of IL-6 induced by P. gingivalis lipopolysaccharide. But compared with the cell control group, the expression levels of IL-6 in the 4 experimental groups were significantly increased, and the expression level of IL-6 was gradually increased with the decrease of the extract concentration. It showed that the pretreatment of osmanthus ethanol extract could effectively inhibit the secretion of IL-6 in a certain extent, but it was still higher than the baseline level.In the same way, the concentration of IL-8 in the supernatant of cell culture was also studied. The results showed that the concentration of IL-8 in the cell control group was 130.47±6.18 pg/mL, and the concentration of IL-8 in the LPS control group was 6350.97±152.01 pg/mL. The concentrations of IL-8 in 4 drug control groups (concentration from high to low) were 40.80±0.87,72.10±1.49,91.03±3.73, 111.00±2.36 pg/mL。The concentrations of IL-8 in the 4 experimental groups were 241.67±12.16,2095.77±191.53,4352.07±99.41,6735.50±17.43 pg/mL. Thus it could be seen that comparing to the cell control group, IL-8 concentrations in LPS control group were significantly higher (P<0.05), indicating that P. gingivalis lipopolysaccharide can successfully induce human periodontal ligament cells to secrete IL-8, mediating periodontal inflammation. Compared with the cell control group, IL-8 concentrations in four drug control group had no significant difference (P>0.05), indicating that pure co-cultured with osmanthus ethanol extract did not have stimulation effect on IL-8 secretion of human periodontal ligament cells. Compared with the LPS group, only at the concentration of 31.25 μg/mL, IL-8 expression level was very low (three concentration, P<0.05), indicating that when the extract concentration is equal to or more than 62.5μg/mL, the pretreatment can inhibit the expression of IL-8 induced by P. gingivalis lipopolysaccharide. But compared with the cell control group, the expression levels of IL-8 in the 4 experimental groups were significantly increased, and the expression level of IL-8 was gradually increased with the decrease of the extract concentration. It showed that the pretreatment of osmanthus ethanol extract could effectively inhibit the secretion of IL-8 in a certain extent, but it was still higher than the baseline level.The fourth chapter:Effect of osmanthus ethanol extract on the expression of oxidative stress markers induced by P. gingivalis lipopolysaccharideThis part of the experiment was to explore the effect of osmanthus ethanol extract at 250μg/mL concentration on the expression of nitric oxide, MDA and superoxide dismutase induced by P. gingivalis lipopolysaccharide by commercial kit, mainly using the human periodontal ligament cells in vitro as the research object.Experimental results showed that in the cells control group, the total nitric oxide content was 2.35±0.33μM/L and in 250μg/mL extract control group, the total nitric oxide content was 2.15±0.27μM/L. In P. gingivalis lipopolysaccharide group, the total nitric oxide content was 8.96±0.47μM/L. In 250μg/mL extract pretreatment group, the total nitric oxide content was 4.69±1.54μM/L. There was no difference in the total content of nitric oxide between the 250μg/mL extract control group and the cell control group (P>0.05). The total content of nitric oxide in the LPS control group was significantly increased and the difference was statistically significant (P<0.05). It suggested that lipopolysaccharide can stimulate the human periodontal ligament cells to synthesize nitric oxide and improve the level of S-nitrosylation. In 250μg/mL extract pretreatment group, the total content of nitric oxide was lower than that in the LPS group, the difference was significant (P<0.05), suggesting that 250μg/mL extract pretreatment can slow down the synthesis of nitric oxide induced by lipopolysaccharide.In the cells control group, MDA content was 0.87±0.19 nmol/mgprot and in 250 μg/mL extract control group, MDA content was 0.98±0.21 nmol/mgprot. In P. gingivalis lipopolysaccharide group, MDA content was 3.12±0.30 nmol/mgprot. In 250μg/mL extract pretreatment group, MDA content was 1.74±0.25 nmol/mgprot. There was no difference in content of MDA between the 250μg/mL extract control group and the cell control group (P>0.05). The content of MDA in the LPS control group was significantly increased and the difference was statistically significant (P<0.05). It suggested that lipopolysaccharide can stimulate the human periodontal ligament cells to synthesize MDA and improve the level of oxidization. In 250μg/mL extract pretreatment group, the content of MDA was lower than that in the LPS group, the difference was significant (P<0.05), suggesting that 250μg/mL extract pretreatment can slow down the synthesis of MDA induced by lipopolysaccharide.In the cells control group, superoxide dismutase content was 40.09±2.34 U/mgprot and in 250μg/mL extract control group, superoxide dismutase content was 42.27±2.57 U/mgprot. In P. gingivalis lipopolysaccharide group, superoxide dismutase content was 22.89±1.98U/mgprot. In 250μg/mL extract pretreatment group, superoxide dismutase content was 35.58±1.67U/mgprot. There was no difference in content of superoxide dismutase between the 250μg/mL extract control group and the cell control group (P>0.05). The content of superoxide dismutase in the LPS control group was significantly decreased and the difference was statistically significant (P<0.05).It suggested that lipopolysaccharide can inhibit the human periodontal ligament cells to synthesize superoxide dismutase and reduce the level of oxidization. In 250μg/mL extract pretreatment group, the content of superoxide dismutase was higher than that in the LPS group, the difference was significant (P<0.05), suggesting that 250μg/mL extract pretreatment can increase the synthesis of superoxide dismutase inhibited by lipopolysaccharide.The experimental results were as follows:● P. gingivalis lipopolysaccharide co-cultured with periodontal ligament cells could make the content of NO increased from 2.35±0.33μM/L to 8.96±0.47 μM/L.250 μg/mL extract pretreatment made NO levels recover to 4.69±0.54 μM/L, significantly decreasing the stimulation of lipopolysaccharide.● P. gingivalis lipopolysaccharide co-cultured with periodontal ligament cells could make the content of MDA increased from 0.87±0.19 nmol/mgprot to 3.12±0.30 nmol/mgprot.250μg/mL extract pretreatment made MDA levels recover to 1.74±0.25 nmol/mgprot, significantly decreasing the stimulation of lipopolysaccharide.● P. gingivalis lipopolysaccharide co-cultured with periodontal ligament cells could make the content of SOD decreased from 40.09±2.34 U/mgprot to 22.89±1.98 U/mgprot.250 μg/mL extract pretreatment made SOD levels recover to 35.58±1.67 U/mgprot, significantly reducing the inhibition of lipopolysaccharide.The fifth chapter:Effect of ethanol extract of Osmanthus fragrans on the expression of protective enzymes and Nrf2 in periodontal ligament cellsIn this study, human periodontal ligament cells were used as the research object, and the effects of osmanthus ethanol extract pretreatment on Nrf2 signal pathway induced by lipopolysaccharide were studied by the intervention of osmanthus ethanol extract and lipopolysaccharide.Experimental results showed that when the expression of HO-1 in the control group, namely pure human periodontal ligament cells group, was set to 1, the expression of HO-1 in P. gingivalis lipopolysaccharide group was 1.33±0.07, in 250 μg/mL osmanthus ethanol extract group was 2.49±0.15. After 250μg/mL osmanthus ethanol extract pretreatment for 2 hours and co-cultured with P. gingivalis lipopolysaccharide and periodontal ligament cells, the content of HO-1 was 3.14±0.20. Thus, comparing to the cell group, lipopolysaccharide and extract co-cultured with human periodontal ligament cells could increase the expression of HO-1 (P<0.05). The osmanthus ethanol extract could stimulate human periodontal ligament cells to produce more content of HO-1 compared with lipopolysaccharide. It suggested that the extract had a synergistic effect with the lipopolysaccharide, which could help the cells to resist the toxic damage induced by lipopolysaccharide.Experimental results also showed that when the expression of NQO1 in the control group, namely pure human periodontal ligament cells group, was set to 1, the expression of NQO1 in P. gingivalis lipopolysaccharide group was 1.71±0.11, in 250 μg/mL osmanthus ethanol extract group was 4.17±0.24. After 250μg/mL osmanthus ethanol extract pretreatment for 2 hours and co-cultured with P. gingivalis lipopolysaccharide and periodontal ligament cells, the content of NQO1 was 5.06±0.38. Thus, comparing to the cell group, lipopolysaccharide and extract co-cultured with human periodontal ligament cells could increase the expression of NQO1 (P<0.05). The osmanthus ethanol extract could stimulate human periodontal ligament cells to produce more content of NQO1 compared with lipopolysaccharide. It suggested that the extract had a synergistic effect with the lipopolysaccharide, which could help the cells to resist the toxic damage induced by lipopolysaccharide.Experimental results showed that when the expression of Nrf2 in the control group, namely pure human periodontal ligament cells group, was set to 1, the expression of Nrf2 in P. gingivalis lipopolysaccharide group was 2.29±0.13, in 250 μg/mL osmanthus ethanol extract group was 3.14±0.18. After 250μg/mL osmanthus ethanol extract pretreatment for 2 hours and co-cultured with P. gingivalis lipopolysaccharide and periodontal ligament cells, the content of Nrf2 was 5.71±0.27. Thus, comparing to the cell group, lipopolysaccharide and extract co-cultured with human periodontal ligament cells could increase the expression of Nrf2 (P<0.05). The osmanthus ethanol extract could stimulate human periodontal ligament cells to produce more content of Nrf2 compared with lipopolysaccharide. It suggested that the extract had a synergistic effect with the lipopolysaccharide, which could help the cells to resist the toxic damage induced by lipopolysaccharide.The experimental results were as follows:● P. gingivalis lipopolysaccharide co-cultured with periodontal ligament cells could make the content of HO-1 increased from 1 to 1.33±0.07.250μg/mL extract pretreatment made HO-1 levels jump to 3.14±0.20, significantly increasing the antioxidant activity of human periodontal ligament cells. Osmanthus ethanol extract and lipopolysaccharide had a synergistic effect, which could help cells to synthesize more content of HO-1 against toxic damage caused by lipopolysaccharide.● P. gingivalis lipopolysaccharide co-cultured with periodontal ligament cells could make the content of NQO1 increased from 1 to 1.71±0.11.250μg/mL extract pretreatment made NQO1 levels jump to 5.06±0.38, significantly increasing the antioxidant activity of human periodontal ligament cells. Osmanthus ethanol extract and lipopolysaccharide had a synergistic effect, which could help cells to synthesize more content of NQO1 against toxic damage caused by lipopolysaccharide.● P. gingivalis lipopolysaccharide co-cultured with periodontal ligament cells could make the content of Nrf2 increased from 1 to 2.29±0.13.250μg/mL extract pretreatment made Nrf2levels jump to 5.71±0.27, significantly increasing the antioxidant activity of human periodontal ligament cells. Osmanthus ethanol extract and lipopolysaccharide had a synergistic effect, which could help cells to synthesize more content of Nrf2 against toxic damage caused by lipopolysaccharide.