Anti-inflammatory Effects And Mechanisms Of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucoside

2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) is an active water solubility compound extracted from the roots of traditional Chinese drug Polygonum multiflorum Thunb. The documents suggested that THSG was a major component of Polygonum multiflorum Thunb pharmacological actions. The documents suggested that Ethanol extracted components of Polygonum multiflorum Thunb inhibited inflammatory responses induced by dimethylbenzene and carrageenin (CGN) and afforded analgesic effects. It had also been reported that THSG afford anti-atherosclerotic and anti-oxidation effect. These effects of THSG may be related to anti-inflammatory effects in experimental animals. The chemical structure of THSG and resveratrol was very similar and belonged to hydroxystilbene compounds. Resveratrol, which extracted from red wine in France, suppressed the activation of cyclooxygenase-2 (COX-2) gene expression and directly inhibits the COX-2 enzyme activity. On the basis of previous work, we wonder whether THSG suppressed COX-2 activity and expression.The following two sections were investigated in this thesis:①Anti-inflammation effects of THSG on experimental animals,②Anti-inflammation mechanisms research of THSG in LPS-stimulated mouse RAW264.7 macrophage cells. To demonstrate these effects of THSG, It provides the theory and experiment documents for developing THSG into a novel drug.PartⅠ: Anti-inflammatory effects THSG on experimental animalsMethods: In mouse ear edema model, after 3 days by oral administration once a day, dimethylbenzene-induced mouse ear edema model was prepared, it was observed to the effet of THSG on mouse ear edema in an hour of dimethylbenzene-induced. In rat paw edema model, after 3 days by oral administration once a day, the CGN-induced rat was determined, it was observed to the effet of THSG on rat paw edema in 1, 2, 4 or 6 hour of CGN-induced. Results:①In mouse edema model, dimetheylbenzene could significantly induce mouse ear edema in 1 hour. The ear edema weight was increased from 7.1±0.7 mg (as control) to 10.7±2.9 mg (P < 0.05). THSG 2.3, 4.6 and 9.2 mg?kg-1 by oral administration could inhibit mouse ear edema and had obvious anti-inflammatory effects in concentration-dependent manner. Edema value was decreased from 4.6±1.5 mg to 3.4±1.0, 1.0±0.4, 0.6±0.2 mg (P < 0.05, P < 0.01). The percentage of inhibition of THSG 9.2 mg?kg-1 was 86.95 % and Indomethacin 13 mg?kg-1, a reference compound, show 89.13 % inhibition.②In rat edema model, CGN could evoke rat paw edema and had obvious effect at 4 or 6 hour. The paw edema circumference was increased from 21.59±0.49 mm to 25.66±1.03 mm (P < 0.01). At 4 hour, THSG 3.2, 6.4 and 12.8 mg?kg-1 could inhibit rat paw edema and had obvious anti-inflammatory effects. Edema value was decreased from 2.60±1.07 mm to 1.47±0.54 mm, 1.56±0.38 mm (P < 0.05), 1.40±0.53 mm (P < 0.01); At 6 hour, THSG 3.2, 6.4 and 12.8 mg?kg-1 could inhibit rat paw edema and had obvious anti-inflammatory effects at dose-dependent manner. Edema value was decreased from 4.07±0.92 mm to 2.28±1.03 mm, 2.02±0.25 mm, 1.81±0.46 mm (P < 0.01); At 6 hour the percentage of inhibition of THSG 12.8 mg?kg-1 was 56 %; Indomethacin 9 mg?kg-1, a reference compound, showed 57 % inhibition in paw edema model.PartⅡ: Anti-inflammation mechanisms of action of THSG in LPS-stimulated mouse RAW264.7 macrophage cells§1 Effects of THSG on cell viabilityMethods: RAW264.7 macrophage cells were determined by MTT assay.Results: RAW264.7 cells were treated with lipopolysaccharide (LPS) 1μg?ml-1 for 6 hours. The viability of cells afforded no change from 100 % to 110.47 % (P > 0.05). After treatment with THSG 1, 10, 100μmol?l-1 for 2 h, no obvious potential effect could be found (LPS + THSG group vs LPS (+)group, P > 0.05) and cell viability was 112.24±7.44 %, 108.49±9.61 %, 108.01±7.01 %, respectively, so THSG had no effects on cell viability. §2 Effects of THSG on PGE2 productionMethods: Prostaglandin E2 (PGE2) production was investigated by Enzyme-Line Immunosorbnent Assay (ELISA).Results: LPS 1μg?ml-1 to RAW264.7 cells for 6 h could change endogenous arachidonic acid (AA) and then significantly increase the production of PGE2 from 44.87±2.48 pg?ml-1 (control level) to 69.07±5.73 pg?ml-1(P < 0.05). THSG 1, 10, 100μmol?l-1 dose-dependently decreased LPS-induced PGE2 production and PGE2 production was 57.87±3.20 pg?ml-1, 38.12±4.73 pg?ml-1, 34.51±3.34 pg?ml-1(P < 0.05, P < 0.01), respectively. THSG 100μmol?l-1 decreased PGE2 production by 50%. NS-398 100μmol?l-1, a selective COX-2 inhibitor was used as a positive control in this assay showed 71% inhibition.§3 Effects of THSG on COX-2 and cyclooxygenase-1 (COX-1) mRNA expressionsMethods: COX-2 and cyclooxygenase-1 (COX-1) mRNA expressions were assayed by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR). Results: LPS 1μg?ml-1 for RAW 264.7 cells for 6 h clearly induced COX-2 mRNA, COX-2/β-actin density ratio exceeded control group from 0.152±0.016 (as control) to 0.734±0.015 (P < 0.01) and COX-1/β-actin density ratio had no effect, compared with control group. LPS 1μg?ml-1 and THSG 1, 10, 100μmol?l-1 for RAW 264.7 cells, COX-2 density ratio was less than LPS group, THSG 1, 10, 100μmol?l-1 could concentration-dependently suppressed COX-2 mRNA expression and density ratio was 0.651±0.021, 0.498±0.016, 0.189±0.016 (P < 0.05, P < 0.01), respectively; THSG 1, 10, 100μmol?l-1 had no effect on COX-1 mRNA and density ratio was almost the same.§4 Effects of THSG on COX-2 and cyclooxygenase-1 (COX-1) protein expressionsMethods: COX-2 and COX-1 protein expression were assayed by Western blot method.Results: LPS 1μg?ml-1after treatment for 6h clearly induced COX-2 protein expression from 65.14±3.6 (as control) to 103.01±9.2 (P < 0.01) and had no effect on COX-1 protein. LPS 1μg?ml-1 and THSG 1, 10, 100μmol?l-1 for RAW 264.7 cells, COX-2 density ratio was less than LPS group, THSG 1, 10, 100μmol?l-1 could concentration-dependently suppressed LPS-stimulated COX-2 protein expression and optical density value is 98.22±5.85, 90.49±8.12, 88.82±7.24 ( P < 0.05, P < 0.01), respectively; THSG 1, 10, 100μmol?l-1 had no effect on COX-1 protein and density value was almost the same.§5 Effects of THSG on COX-2 enzyme activityMethods: COX-2 enzyme activity was investigated by ELISA.Results: COX-2 enzyme catalyzed exogenous AA; metabolite PGE2 production could represent COX-2 enzyme activity. PGE2 production was much and enzyme activity could be high. LPS 1μg?ml-1for RAW264.7 cells for 6 hour dramatically increased PGE2 production from 59.25±8.17 pg?ml-1 (the basal level) to 79.95±5.53 pg?ml-1 (1×105 cells in a 96-well plate, P<0.05). THSG 1, 10, 100μmol?l-1 could concentration -dependently decreased PGE2 production from AA and PGE2 production was decreased from 79.95±5.53 pg?ml-1 to 66.32±1.83 pg?ml-1, 48.13±4.65 pg?ml-1, 34.78±0.96 pg?ml-1 (P< 0.05, P< 0.01), respectively. The percentage of inhibition of THSG or NS-398 100μmol?l-1 was 56 % and 68 %, respectively.Conclusion:①THSG could afford anti-inflammatory effect in mouse acute inflammatory model, this effect may be related to inhibit mouse ear capillary vessel edema.②THSG could afford anti-inflammatory effect in rat inflammatory model, this effect may be related to inhibit inflammation mediator (prostaglandins, 5-hydroxytryptamine).③THSG 100μmol?l-1 did not show any cytotoxicity judged by MTT assay, indicating that the inhibition of PGE2 production by THSG was not associated with its cytotoxicity.④THSG could dose-dependently reduce the PGE2 production, from endogenous AA in LPS-activated RAW 264.7 cells, indicating that THSG could directly decrease PGE2 production, this effect was related to inhibit COX-2 gene expression or directly inhibit COX-2 enzyme activity.