| 1 Objective Copper pollution in current environment is more and more serious,while copper is an essential trace element in the human body and play a crucial role in the life activities,but the influence of high concentration of copper accumulation in the body is still not attracted enough attention,especially the influence on female endocrine and follicular development of ovarian function is still poorly known.In response to this phenomenon,this subject will continue the previous research,to study the effect of copper load on human ovarian function and explore effective methods of Chinese medicine treatment.This topic will be following the previous experiment,mainly through animal experiments in mice and human granulosa cells in vitro experiment,from the molecular and cellular level to understand the effect of copper load on apoptosis of ovarian granulosa cell and its influence on the mechanism of ERK1/2 signaling pathway and the mechanism of traditional Chinese medicine of tonifying kidney,removing blood stasis and eliminating phlegm,in order to provide theoretical basis for the female who have endocrine disorders and follicular developmental disorders such diseases related to ovarian function caused by copper load,also let people pay attention to copper pollution effects on ovarian function of women.2 Methods2.1 animal experiments in mice30 female mice were randomly divided into three groups:Copper load model group/tonifying kidney removing blood stasis eliminating phlegm model group/control group,ten rats in each group.Fill the stomach of the mice of copper load model group and tonifying kidney removing blood stasis eliminating phlegm model group by copper concentration of 100 mg/kg body weight,the volume of lavage is 0.2ml solution for every 10 g weight every day;Fill the stomach of the mice of the control group with the same volume of saline;are all the same time every morning to fill the stomach once.On the fifteenth day,the mice were given intragastric administration,dosage of 0.2ml solution for every 10 g body weight in mice,are all the same time every afternoon to fill the stomach once.Fill the stomach of the mice of copper load model group and control group with the same volume of saline,to the end of twenty-eighth days of copper.At the corresponding time point,the mice were killed and the bilateral ovaries were Individually treated 。 To observe the apoptosis of granulosa cells by tunel method;To detect ERK1/2 and P-ERK1/2 and observe the level of ERK1/2 phosphorylation using Western blot method.2.2 human granulosa cells in vitro experiment The human granulosa cells in vitro cultured are divided into three groups:control group/copper load group/tonifying kidney removing blood stasis eliminating phlegm model group/U0126 group,culture corresponding treatment.To observe the proliferation of granulosa cells using ki67 staining To observe the apoptosis of granulosa cells by tunel method;To detect ERK1/2 and P-ERK1/2 and observe the level of ERK1/2 phosphorylation using Western blot method.3 Results3.1 the result of animal experiments in mice Compared with the normal control group,the number of apoptotic cells in the ovarian granulosa cells of copper load model group increased and there was significant difference;Compared with the copper load model group,the number of apoptotic cells in the ovarian granulosa cells of tonifying kidney removing blood stasis eliminating phlegm model group decreased and there was significant difference.Compared with the normal control group,the expression of P-ERK1/2 in the copper load model group was significantly lower than that in the control group;Compared with the copper load model group,the expression of P-ERK1/2 in the tonifying kidney removing blood stasis eliminating phlegm model group was significantly higher than that in the copper load model group.3.2 the result of human granulosa cells in vitro experiment Compared with the normal control group,the number of proliferative human granulosa cells of copper load group decreased,the number of apoptotic human granulosa cells increased and there was significant difference;Compared with the copper load group,the number of proliferative human granulosa cells of tonifying kidney removing blood stasis eliminating phlegm model group increased,the number of apoptotic human granulosa cells decreased and there was significant difference;Compared with the other groups,the number of proliferative human granulosa cells of U0126 group decreased,the number of apoptotic human granulosa cells increased and there was significant difference.Compared with the normal control group,the expression of P-ERK1/2 in the copper load group of human granulosa cells was significantly lower than that in the control group;Compared with the copper load group,the expression of P-ERK1/2 in the tonifying kidney removing blood stasis eliminating phlegm group of human granulosa cells was significantly higher than that in the copper load model group.4 conclusion4.1 Copper loading can induce apoptosis of ovarian granulosa cells;4.2 Traditional Chinese medicine of tonifying kidney,removing blood stasis and eliminating phlegm can abate the apoptosis of ovarian granulosa cells loaded with copper;4.3 Copper loading can inhibit the phosphorylation of ERK1/2 in ovarian granulosa cells;4.4 Traditional Chinese medicine of tonifying kidney,removing blood stasis and eliminating phlegm can induce the phosphorylation of ERK1/2 in ovarian granulosa cells inhibited by copper loading;4.5 Copper loading can inhibit the phosphorylation of ERK1/2 in ovarian granulosa cells,and then promote the apoptosis of granulosa cells,and then affect the growth and development of ovarian follicles and endocrine function of the ovary;4.6 Traditional Chinese medicine of tonifying kidney,removing blood stasis and eliminating phlegm can induce the phosphorylation of ERK1/2 in ovarian granulosa cells inhibited by copper loading,and then abate the apoptosis of ovarian granulosa cells loaded with copper,and then affect the growth and development of ovarian follicles and endocrine function of the copper loaded ovary. |
