| 1 ObjectiveThe current environment of copper pollution increase sharply. Copper is an essential trace element for human body, which plays an important role in the metabolic processes. But a high concentration of copper and its effect on the body is not yet clear,especially in follicular development and the female reproductive tract which is lack of systematic research. For this situation, the research is proposed to observe and study the poisoning of copper effect on human reproduction and follicle development, and explore the effective treatment of traditional Chinese medicine. The goal of this research is to investigate the disorders of menstrual cycle in Wilson disease patients who are human natural copper poisoning model, the effect of invigorating kidney,eliminating phlegm and removing blood stasis recipe on follicular development and menstrual disorder in Wilson disease patients, and a preliminary exploration of the mechanism of copper on follicular development in the animal model and the action of traditional Chinese medicine.2 Methods2.1 Clinical investigationAll patients included had confirmed diagnosis of Wilson’s disease,attended our clinic of The Encephalopathy Center of the First Affiliated Hospital of Anhui University of Chinese Medicine from May 2015 to October 2015, and all of them were female patients of childbearing age A retrospective questionnaire was used to survey they menstrual history(age at menarche, menstrual cycle, the number of days passing,menstrual flow, etc.).2. 2 Clinical research40 cases of Wilson’s disease patients which randomly choosed from The Encephalopathy Center of the First Affiliated Hospital of Anhui University of Chinese Medicine from September 2014 to August 2015. They were randomly divided into observer group(20 cases), control group(20 cases). Control group taked regular decoppering therapy and the observer group not only taked regular decoppering therapy, but also received the supplement treatment of invigorating kidney, eliminating phlegm and removing blood stasis recipe, taking for three menstrual cycles. To observe changes of the diameter of the dominant follicle and delayed menstrual cycle of those patients.2.3 Experimental research60 ICR female mice were randomly divided into those three groups: normal control group, copper load model group, invigorating kidney, eliminating phlegm and removing blood stasis recipe group,n = 20. The mices of copper load model group and invigorating kidney, eliminating phlegm and removing blood stasis recipe group were gavaged with 0.2ml copper solution / 10 g body weight daily, the dose of the copper solution is copper concentration of 100 mg / kg of body weight,the mices of normal control group were gavaged with an equal volume of saline daily. On these mices taking for 15 days, according to body surface area conversion method, the dose of invigorating kidney, eliminating phlegm and removing blood stasis recipe for adults converted into mice, gavage dose: 0.2ml liquid / 10 g body weight, daily afternoon 1time. Normal control group and copper load group were gavaged with an equal volume of saline, until to the end of the 28 th day to gavage with copper. The morphological changes of cells in mouse ovaries were observed by HE staining; Ultrastructure of the ovarian follicles and granulosa cells had been studied with the electron microscope;Observe the developmental potential of those mices by parthenogenetic activation oocytes; Analysis of p-ERK and ERK levels.3 Results3.1 Menstrual disorders in Wilson’s disease patientsIn the 109 patients, 2 patients with primary amenorrhea, 5 patients with secondary amenorrhea. In the 107 cases of patients with menstrual history, the 62 menstrual disorders.3.2 The changes of number and the size of dominant follicle, and curative effect in Wilson’s disease patientsAfter treatment, in both of the treatment group and the control group, the number of well developed follicles which is > 15 mm in diameter significantly more than before the treatment, the diameter of the dominant follicle significantly greater than before treatment(P < 0.05). The size of the well developed follicles and the number of dominant follicle in the treatment group were significantly better than that in control group(P < 0.05 or 0.05). Efficiency of the treatment group was obviously higher than that of control group, compared between two groups, difference has statistical significance(P < 0.001).3.3 Experimental results3.3.1 The morphological changes of ovary by copper load in mice and the therapeutic effect of invigorating kidney, eliminating phlegm and removing blood stasis recipe Compared with normal control group, the number of normal secondary follicles and normal sinus follicles in copper load model group mices were significantly reduced(P<0.01), the number of atresic secondary follicle and atresic sinus follicle were significantly increased(P< 0.01). Compared with copper load model group,invigorating kidney, eliminating phlegm and removing blood stasis recipe group can increase the number of normal sinus follicle and secondary follicular, reduce the number of atresic sinus follicle and atresic secondary follicular(P < 0.05).3.3.2 The impact of copper load on oocytes ultrastructure and the therapeutic effect of invigorating kidney, eliminating phlegm and removing blood stasis recipeCompared with normal control group, the number of apoptotic granulosa cells and abnormal degeneration of mitochondria in copper load model group mices were significantly increased(P< 0.01). Compared with copper load model group,invigorating kidney, eliminating phlegm and removing blood stasis recipe group can increase the number of normal mitochondria(P < 0.05).3.3.3 The effects of copper load on oocyte development potential and the therapeutic effect of invigorating kidney, eliminating phlegm and removing blood stasis recipe Compared with normal control group, the rate of blastocyst formation in copper load model group is significantly reduced(P< 0.01). Compared with copper load model group, invigorating kidney, eliminating phlegm and removing blood stasis recipe group can significant increase rate of blastocyst formation(P < 0.05).3.3.4 The inhibitory effect of copper load on oocytes ERK1 / 2 and the activation function of invigorating kidney, eliminating phlegm and removing blood stasis recipe,with kidney and phlegm and remove blood stasisCompared with normal control group, the level of ERK1 /2 phosphorylated proteins relative expression significantly reduced in copper load model group(P < 0.01).Compared with copper load model group, invigorating kidney, eliminating phlegm and removing blood stasis recipe group can significant increase the level of ERK1 /2phosphorylated proteins relative expression(P < 0.05).4 conclusion4.1 Menstrual disorder is a common phenomenon among Wilson’s disease patients of childbearing age, but the mechanism is not clear yet, more research is needed;4.2 Invigorating kidney, eliminating phlegm and removing blood stasis recipe can obviously promote the follicular development in Wilson’s disease patients,significantly improve the delayed menstrual cycle in patients with Wilson’s disease;4.3 Copper can load damage to the structure of ovaries, interfere with the follicular development and mature, decend the development potential of oocyte;4.4 Invigorating kidney, eliminating phlegm and removing blood stasis recipe can to some extent improve the damage which is caused by copper load;4.5 Copper load could inhibit the phosphorylation of Erk1/2 of mice oocyte, and invigorating kidney, eliminating phlegm and removing blood stasis recipe could stimulate the phosphorylation of Erk1/2 of copper load mice oocyte;4.6 Invigorating kidney, eliminating phlegm and removing blood stasis recipe may via stimulating the phosphorylation of Erk1/2 of copper load mice oocyte to improving the development of follicular oocytes and their potential. |
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