Study The Inhibitory Effect Of Genipin On The Inflammatory Response Of KGN Cell Line Induced By LPS

Objective:To study the effects of different concentrations of Genipin on the secretion function and regulation pathway of KGN cell line under inflammatory and non-inflammatory conditions;To demonstrate the anti-inflammatory effect of Genipin,and discussion the possible regulatory pathway of Genipin in the anti-inflammatory function.Methods:1. Cck-8 was used to detect the growth of human ovarian granule cell line?KGN?with different concentrations of Genipin:After 24 hours of culture,different concentrations of Genipin were added,respectively:0?0.05mmol/L?0.1mmol/L?0.2mmol/L?0.3mmol/L.OD value of KGN cell line with different concentration was measured at 0h?12h?24h?48h.2.Effects of different concentrations of Genipin on KGN cell line in non-inflammatory condition:After 24 hours of culture,different concentrations of Genipin were added,respectively:0?0.05mmol/L?0.1mmol/L?0.2mmol/L?0.3mmol/L.Elisa detected the secretion of E₂?P?IL-4?IL-5 after 24h of cultured,Protein was extracted from the bottom cells and the expression of UCP2?17?-HSD2?CYP17A1?CYP19A1 protein was detected by Western blot.3.Effects of different concentrations of Genipin on the inflammatory responsing to KGN cell line induced by LPS:1)KGN cell line intervened by 2ug/ml LPS,to set the oxidative stress condition;2)Effects of Genipin on secretion function and regulation pathway of KGN cell line in inflammatory condition:cultured after 24 hours,different concentrations of Ginipin were added,respectively:0?0.05mmol/L?0.1mmol/L?0.2mmol/L?0.3mmol/L.Elisa detected the secretion of E₂?P?IL-4?IL-5.Protein was extracted from the bottom cells and the expression of UCP2?17?-HSD2?CYP17A1?CYP19A1?NF-?B protein was detected by Western blot.Results:1. OD value decreased gradually after high concentration of Genipin??0.1mmol/L?were used to interfere the KGN cell line,and the difference was significant?P<0.05?,The OD value decreased with the increase of the concentration of Genipin;OD values at low concentration of Genipin were not significantly changed compared with those of control group?P>0.05?,and there was no significant difference?P>0.05?.2. Effects of different concentrations of Genipin on KGN cell line in non-inflammatory condition:With the increase of Genipin concentration,the secretion of E₂?P?IL-4?IL-5 in KGN cell line decreased gradually,and the difference all were significant?P<0.05?.With the increased of Genipin concentration,UCP2 protein engenderly reduced contrasting with the control group,and the difference was significant?P<0.05?.CYP17A1 protein expression increased gradually compared with the control group,the change was significant?P<0.05?;CYP19A1 and 17?-HSD2protein expression levels of the experimental group almost as the same as the control group,but there was no significant difference?P>0.05?.3. Study the effects of different concentrations of Genipin on the inflammatory response to KGN cell line induced by LPS:1)In oxidative stress condition,E₂secretion gradually decreased with the increase of Genipin concentration,but the change between the experimental group and the control group were not significant?P>0.05?.At high concentrations of Genipin??0.1mmol/L?,Different secretion of progesterone between the experimental group and the blank group was statistically significant?P<0.05?.2)As for IL-5,statistically higher compared with the blank group,the difference was significant?P<0.05?,The density in the experimental group cut down significantly compared with the control group,and the variation was statistically significant?P<0.05?.3)The experimental group of IL-4 decreased gradually when contrasted with the control group,Among them,0.05mmol/L and 0.3mmol/L gradually declined contrasting with the control group,with statistically significant change?P<0.05?.4)The experimental group of CYP17A1 protein decreased gradually contrasted with the control group,the transformation was statistically significant?P<0.05?,in the experimental group,both of the two protein increase than the control group,but the mutation was not statistically significant?P>0.05?.The experimental group of CYP19A1 protein almost as same as the control group and the blank group,the difference were not significant?P>0.05?.5)The experimental group of 17?-HSD2 was more than the control group,but there was no change in protein expression between them.6)UCP2 protein expression in the control group more than the blank group,The variation was statistically significant?P<0.05?.the experimental group less than the control group,but the variation was not t significant?P>0.05?.7)NF-?B protein expression in the control group more than the blank group,and NF-?B protein expression in the experimental group less than the control group,excepting the 0.05mmol/L group,The 0.1mmol/L group was declined markedly,and the variation was not significant?P<0.05?.Conclusion:1.In this experiment,different concentrations of Genipin were used to interfere with the KGN cell line,and the inhibition was obvious when the concentration of Genipin is equal or greater than 0.2mmol/L,As the concentration of Genipin increased,The inhibitory effect was more obvious.2.With different concentrations of Genipin were used to interfere with the KGN cell line,The secretion of IL-4?IL-5?UCP2 were decreased,it suggested that Genipin has anti-inflammatory effect on KGN cell line under inflammatory stress,UCP2 may be one of its anti-inflammatory regulatory proteins;Excepting the regulated by key enzymes,UCP2 may play a regulatory role in the changes of estrogen and progesterone levels in the body under non-inflammatory conditions.3. Effects of Genipin on the inflammatory response of KGN cell line induced by LPS.The expression of IL-4?IL-5?UCP2 and NF-?B increased under oxidation condition,It was proved that the model of oxidative stress induced by LPS in KGN cell line was successful.under the oxidative stress,the inflammatory secretion index and protein were decreased after Genipin interfered,It is suggested that Genipin has an anti-inflammatory effect on KGN cell line under oxidative stress,and its anti-inflammatory effect may be regulated by NF-?B and UCP2 pathway.4. In this experiment,the effect of Genipin on KGN cell line under oxidative stress was preliminarily explored,which provided the preliminary experimental basis for the subsequent research on Genipin in the female reproductive system.