| Objective:This experiment is based on the theoretical compatibility of the classic "jealous infertility" theory of traditional Chinese medicine and the psychological stress theory of modern medicine.By preparing models of psychological stress in mice,detecting the mitochondrial function of mouse oocytes and the level of apoptotic factors in oocytes,etc,this experiment explores the effects of increased levels of a ctive reactive oxygen species in the body caused by psychological stress on the mitochondrial function of mouse oocytes,and then affects the apoptosis level of mouse oocytes.This paper provides scientific basis for the theory of liver depression causing female infertility which is included in the classic "jealous infertility" theory of traditional Chinese medicine.Method: Experiment A : Effects of psychological stress on mitochondrial function in mouse oocytesPrepare mouse models of strong,medium,and weak psychological stress,take oocytes from each group,then use real-time quantitative PCR to detect mt DNA copy number,use MitoTracker Green(mitochondrial green fluorescent probe)to detect mitochondrial distribution,use Biofluorescence technology to detect the ATP level of mouse oocytes in each group,and use enzyme immunoassay to detect the expression level of 8-OHd G in oocytes.So that to research the effects of psychological stress on mitochondrial function in mouse oocytes Experiment B : Effect of psychological stress on the level of mouse oocyte apoptosisUse RT-PCR and Western blot to detect Bcl-2,Bax,Cyt C,and Caspase-3 apoptotic pathway factor levels,and use TUNEL(terminal deoxynucleotidyl transferase d UTP nick end labeling)to determine the oocyte apoptosis rate.So that to research the effect of psychological stress on the level of mouse oocyte apoptosis.Result:The experimental data was analyzed and processed by SPSS21.0 statistical software.Oneway analysis of variance was used to compare measurement data between multiple groups.For further comparisons between groups,if the variances were equal,the LSD(Fisher'sleastsignificant difference test)test was used.If the variances were not the same,Dunnett's T3 test was used.The difference was statistically significant at P<0.05.Experiment A 1.Mitochondrial mt DNA copy number in mouse oocytes of each groupThe copy number of mitochondrial mt DNA in oocytes of mice with strong,medium and weak stimulation was lower than that in the normal group.A further pairwise comparison showed that compared with the normal group,the mt DNA copy number in oocytes of the strong and medium stimulation group was decreased,and the difference was statistically significant(P <0.05).Compared with the strong stimulation group,the mt DNA copy number in oocytes in mice in the medium and low stimulation groups increased,and the difference was statistically significant(P <0.05).Compared with the medium stimulation group,the mt DNA copy number in oocytes of mice in the weakly stimulation group increased,and the difference was statistically significant(P <0.05).2.Mitochondrial distribution of mice in each groupWith the increase of stimulation intensity,the uneven distribution of mitochondria in oocytes in each group increased,and the distribution of mitochondria in oocytes in normal mice was evenly distributed.The distribution of mitochondria in oocytes in mice in strong stimulation group was significantly shifted to one side.The oocyte mitochondria were distributed in the center and edge of the cytoplasm.The oocyte mitochondria distribution in mice in the weakly stimulated group was not significantly different from that in the normal group.3.ATP levels of mouse oocytes in each groupThe concentration of ATP in the mouse oocytes in strong,medium and weak stimulation groups were lower than those in the normal group,and the difference was statistically significant(P <0.05).Further pairwise comparisons showed that with the increase of the stimulation intensity,the concentration of ATP in mouse oocytes decreased,and the differences were all statistically significant(P <0.05).4.Enzyme immunoassay to detect 8-OHd G expression in oocytesThe level of 8-OHd G expression in mouse oocytes in strong,medium and weak stimulation groups was higher than that of the normal group,and the difference was statistically significant(P <0.05).Further pairwise comparison showed that with the increase of stimulation intensity,the expression level of 8-OHd G in mouse oocytes increased,and the differences were statistically significant(P <0.05).Experiment B 1.Levels of apoptotic pathway factors Bcl-2,Bax,Cytc,Caspase-3 in mouse oocytes of each groupThe levels of anti-apoptotic factor Bcl-2 in mouse ocytes with strong,moderate and weak psychological stress were lower than those in the normal group,and the difference was statistically significant(P <0.05);further Pairwise comparison showed that with the increase of stimulation intensity,the levels of anti-apoptotic factor Bcl-2 in mouse oocytes decreased,and the differences were statistically significant(P <0.05);The levels of pro-apoptotic factors Bax,Cytc,and Caspase-3 in mouse ocytes with strong,moderate and weak psychological stress were higher than those in the normal group,and the difference was statistically significant(P <0.05);further Pairwise comparison showed that with the increase of stimulation intensity,the levels of pro-apoptotic factors Bax,Cytc,and Caspase-3 in mouse oocytes increased,and the differences were statistically significant(P <0.05).2.Level of apoptosis in mouse oocytes in each groupWith the increase of the intensity of psychological stress stimulation,the mouse oocyte apoptosis rate gradually increased.Compared with the normal group,the mouse oocyte apoptosis rate in the strong stimulation group increased significantly and there was a statistical difference(P <0.05).).The mouse oocyte apoptosis rate in the medium stimulation group and the weak stimulation group increased,but there was no statistical difference compared with the normal group.Compared with the strong stimulation group,The mouse oocyte apoptosis rate in the medium and weak stimulation group decreased,and there were statistical differences(P <0.05).Compared with the medium stimulation group,the mouse oocyte apoptosis rate in the weak stimulation group was lower,but there was no statistical difference between the two.Conclusion:1.Psychological stress can affect the mitochondrial function of oocytes,leading to a decrease in mt DNA copy number of mitochondria,a decrease in intracellular ATP content,an uneven distribution of mitochondria within the cell,and an increase in the level of DNA oxidation damage marker 8-OHd G;and the stronger the level of psychological stress stimulation,the more severe the damage to mitochondrial function.As the most important organelle that powers oocytes,the damage of mitochondria can directly lead to the decline of oocyte quality,affect ovarian function,and cause female infertility.2.Psychological stress can lead to an increase in the rate of oocyte apoptosis,an increase in the levels of pro-apoptotic factors in the oocytes,and a decrease in the levels of anti-apoptotic factors;and the more severe the level of psychological stress,the higher the expression level of pro-apoptotic factors in the oocytes,the lower the expression of anti-apoptotic factors,the higher the level of oocyte apoptosis,and the more obvious the decline of ovarian reserve function.3.Psychological stress is similar to the theory of traditional Chinese medicine liver depression in terms of etiology and clinical symptoms.Psychological stress leads to an increase in the apoptosis rate of oocytes and a decline in ovarian reserve function by affecting the energy supply of mitochondria in oocytes.Spleen suppression and splenic primary dysfunction cause the acquired essence and innate essence to be difficult to complement each other,which in turn affects the kidney's reproductive function with a high degree of uniformity.Through further experimental verification,it provides modern medical theoretical basis for the traditional Chinese medicine to relieve liver and spleen,and kidney seed method. |
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