| Objective:To explore the effect of artemisitene(AT)on triptolide(T9)induced ovarian toxicity and granulosa cell apoptosis at cell and molecular levels;and to explore the method of ingredient preparation.Artemsia annua L.extract fraction n1(AE-n1)with high content of AT was prepared to reduce the ovarian toxicity of Tripterygium glycosides,enrich the biological activity of artemisinin,and provide data support for the detoxification study of TG.Methods:1.The effect of AT on premature ovarian insufficiency(POI)model induced by T9 in miceThe SPF grade adult BALB/c female mice were randomly divided into 6 groups:control group,model group,AT low dose group,AT middle dose group,AT high dose group and positive drug group.T9(0.5 mg/kg/day)was injected intraperitoneally for 28 days to establish a mouse POI model.At the same time,low,middle and high doses of AT and 17 β-estradiol(E2)were injected intraperitoneally.During the experiment,the behavioral changes,the amount of food and water intake,the weight and the estrus cycle changes were observed.At the end of the experiment,the mice were sacrificed,the serum was collected,serum sex hormone indexes Luteinizing hormone(LH)、Follicle stimulating hormone(FSH)、Estradiol(E2)、Anti-Mullerian hormone(AMH)were detected by ELISA assay,the ovary and uterus were observed,the weight of ovary and uterus was weighed and organ index was calculated,part of the ovary was dissected and embedded by paraffin and sectioned,histopathological changes were observed after HE staining,and the apoptosis of ovarian cells were observed after TUNEL fluorescence staining,and part of the ovary was examined by transmission electron microscopy to observe the ultrastructural changes.The above methods were used to evaluate the protective effect of AT on T9-induced POI mice.2.The effect of AT on the apoptosis of primary rat ovarian granulosa cells induced by T9The effect of AT on the apoptosis of rat ovarian granulosa cells induced by T9 was studied in vitro.Female SD immature rats were injected with 40 IU pregnant mare serum gonadotropin(PMSG)intraperitoneally.48 hours later,the primary granulosa cells were isolated and cultured.The granulosa cells were identified by follicle-stimulating hormone receptor(FSHR)immunofluorescence staining method.After culture and passage,CCK-8 method was used to detect the effect of AT and T9 on KGN cell viability,the IC50 of T9 was calculated,the effective dose of AT was explored,and apoptosis rate was detected by flow cytometry method.3.Study on the molecular mechanism of AT against T9-induced apoptosis of ovarian granulosa cells based on KGN cellsTo explore the molecular mechanism of AT antagonizing the apoptosis of ovarian granulosa cells induced by T9,KGN cells were resuscitated for culture,and the cells in logarithmic growth period were seeded into the culture plate.CCK-8 kit was used to detect the effect of AT and T9 on the activity of KGN cells,and IC50 of T9 was calculated,and the effective dose of AT was explored.Following AT and T9 intervention,the apoptosis rate of KGN cells was detected by Annexin V-FITC/PI reagent.The expression changes of apoptosis-related proteins were observed by Western Blot.Tne small interfering RNA(siRNA)transient transfection technology was used for inducing gene silencing in cells to verify the related signal pathway.4.Monitoring the binding ability of AT,T9 with tumor necrosis factor receptor 1(TNF-R1)protein by biolayer interferometryThe affinity of AT and T9 with TNF-R1 was monitored by biolayer interferometry.Firstly,TNF-R1 protein was labeled with biotin and the SSA sensor was solidified,and verify whether the label with biotin was succeed.In the concentration gradient range of AT,T9 10 nM,100 nM,1000 nM,10 μM,100 μM,1000 μM,the concentration range with high affinity was chosen,and then their affinity with TNF-R1 protein was monitored in this range of concentration.Finally,the results were analyzed by Fortebio analysis software,the binding constant Kon and dissociation constant Kdis were calculated,and finally the affinity constant KD value was obtained.5.Preparation and quality control of AE-n1Artemisia annua L.extract was extracted and separated by column chromatography to remove the superfluous impurities,then AT was added according to the method of component distribution ratio to prepare the components with higher AT content and establish the quality control standard.6.Effect of AE-nl in POI rat model induced by TGSPF grade female adult SD rats were randomly divided into 6 groups:control group,model group,AE-n1 low dose group,AE-n1 middle dose group,AE-n1 high dose group and positive drug group,with 8 rats in each group.TG 75 mg/kg/day was gavaged for 28 days to establish the POI rat model,At the same time,the low,middle and high doses of AE-nl and estradiol valerate(EV)were given by gavage.During the experiment,the changes of behavior,food and water consumption,body weight and estrus cycle were observed.At the end of the experiment,the rats were sacrificed,serum was collected,serum sex hormone indexes LH,FSH,E2 and AMH were detected by ELISA method;the ovary and uterus were observed;the weight of ovary and uterus was weighed,and organ and uterus index was calculated;part of ovary was dissected and embedded with paraffin and sectioned,histopathological changes were observed by HE staining,TUNEL fluorescence staining were used to observe the apoptosis of ovarian cells.Part of ovary was used to observe the ultrastructural changes of ovary by transmission electron microscopy.Part of ovarian tissues was used to detect the expression of apoptosis-related proteins.The spleen of rats was dissected and the spleen cells were extracted.The proportion of Th17,Treg cells in the spleen was detected by flow cytometry and the ratio of Th17/Treg was observed.The above methods were used to evaluate the protective effect of AE-nl on the ovaries of POI rats induced by TG.Results:1.AT could antagonize the ovarian toxicity of T9,improve the disease state of POI rats induced by T9,increase the amount of food and water intake and weight of mice,reduce the ovarian index and uterine index,reduce the apoptosis of ovarian granulosa cells,improve the pathological state of ovary,promote the development of follicles,reduce the serum levels of LH and FSH,and increase the serum levels of E2 and AMH,the estrous cycle of mice was restored to a certain extent.2.In vitro,140 nM T9 showed significant inhibition on the activity of rat primary ovarian granulosa cells,while 0.5 μM,1 μM,2 μM three concentrations of AT can antagonize the damage of T9 on primary ovarian granulosa cells.Flow cytometry showed that the three concentrations of artemisinin can reduce the apoptosis of granulosa cells induced by T9.3.In human ovarian granulosa cell lines KGN cells,300 nM T9 could significantly inhibit the activity of KGN cells,while the activity of KGN cells could be increased by 0.25 μM,0.5 μM and 1 μM AT.The results of flow cytometry showed that the three concentrations of AT could reverse the increase of apoptosis rate of granulosa cells induced by T9.Further study showed that AT antagonized the apoptosis of KGN cells induced by T9 through TNF-Rl-mediated apoptosis pathway4.The affinity constant of AT and TNF-R1 was KD=3.46×10-4 M,and that of T9 and TNF-R1 was KD=2.82×10-4 M.5.AE-n1 with higher AT was prepared.6.AE-n1 can reduce the toxicity of TG to ovary,improve the disease state of POI caused by TG in SD rats,increase the amount of food and water intake and weight of mice,reduce the ovarian index and uterine index,reduce the apoptosis of ovarian granulosa cells,improve the pathological state of ovary,promote the development of follicles,reduce the level of serum LH and FSH,and elevate serum E2 and AMH levels.It can also improve the imbalance of Th17/Treg immune cells and restore the estrous cycle to a certain extent.AE-n1 could inhibit the toxic effect of TG on rat ovary via AE-nl-mediated apoptosis pathway.Conclusion:1..AT could reduce T9 induced ovarian toxicity and granulosa cell apoptosis through TNF-R1 mediated exogenous and mitochondrial apoptosis pathway.2.AE-n1 with high AT content prepared by ingredient preparation method could antagonize the ovarian toxicity of TG,which may be related to the inhibition of TNF-R1-mediated exogenous and mitochondrial apoptosis. |
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