Effect Of Soothing Liver And Tonifying Kidney On Apoptosis Of Granulosa Cells In Rats With Premature Ovarian Failure Based On Oxidative Stress

ObjectiveThe rat model of liver depression and kidney deficiency syndrome of premature ovarian failure（POF）was established by cyclophosphamide（CTX）+chronic unpredictable mild stress（CUMS）method.The molecular basis of liver depression and kidney deficiency syndrome of POF and the way of improving POF by regulating ovarian granulosa cell apoptosis by oxidative stress were discussed.MethodsA total of 52 SPF female SD rats aged 8 weeks were divided into normal group（10 rats）and model group（42 rats）by random number table method.POF model rats were prepared by intraperitoneal injection of cyclophosphamide for 15 consecutive days in the model group.Meanwhile,chronic unpredictable mild stress method was used to establish liver depression model for 21 consecutive days.Two rats were randomly selected from each group to observe the body weight,estrous cycle,behavioral experiment and ovarian histomorphology to determine the success of modeling.The rats were randomly divided into model group,low-dose group,medium-dose group,high-dose group and estrogen group,with 8 rats in each group.The intragaural doses of Chaihu Shugan SAN combined with Zuogui Yin in low-dose,medium-dose and high-dose groups were 3.39g/kg,6.77g/kg and 13.55g/kg,respectively.The estrogen group was intragastrically administrated with estradiol valerate（bugalol）0.105mg/kg,and the normal group and model group were intragastrically administrated with equal volume of sterilized water,and the intervention lasted for 3 weeks.Hematoxylin-eosin（HE）staining was used to observe the morphological changes of ovarian tissue.The contents of follicle-stimulating hormone（FSH）,estradiol（E₂）,anti-Muller-tube hormone（AMH）and statin B（INHB）in serum were detected by enzyme-linked immunosorbent assay（ELISA）.The apoptosis rate of ovarian granulosa cells was detected by d UTP notch end labeling（TUNEL）mediated by terminal deoxynucleotide transferase.The m RNA expressions of Bax,Bcl-2,ROS and SOD-2 were detected by real-time quantitative PCR（q-PCR）.The levels of ROS in ovarian tissues were detected by chemical fluorescence.The protein expression levels of Bax,Bcl-2 and SOD-2 in rat ovarian tissue were detected by Western blotting（WB）.Results1.Comparison of indicators after molding:After the modeling,compared with the normal group,the body weight of rats in the model group was decreased,the estrous cycle was disturbed,the total distance of open field movement,the central area residence time and the number of upright times were significantly reduced,and the sucrose water consumption rate was decreased.Under the optical microscope,the ovarian tissue structure of the model group was not clear,the follicles dilated obviously,follicles of all levels were rare,and the granulosa cell layer became thin.2.Comparison of indicators after intervention:（1）Comparison of ovarian tissue anatomy and morphology:under stereopicroscope,the ovary surface of rats in the normal group was bright red with uniform particles.The ovary tissue of model group was pale and its envelope was thickened.Compared with the model group,the ovary surface color of rats in low,medium and high dose groups and estrogen groups was rudder.Under optical microscope,follicles at different stages of development were observed in the normal group.The granulosa cell layer was thick and closely arranged,and there were more mature follicles.In the model group,the ovarian tissue structure was not clear,the granulosa cell layer was obviously thinner,and multiple vacuolar follicles could be seen.Compared with the model group,the number of follicles increased in the low,medium and high dose groups and estrogen groups,and the granulosa cell layer was thickened.（2）Comparison of serum FSH,E₂,AMH and INHB levels:Compared with normal group,serum FSH level of rats in model group was significantly increased（P<0.01）,while serum E₂,AMH and INHB levels were significantly decreased（P<0.01）;Compared with model group,serum FSH levels in low,medium and high dose groups and estrogen groups were significantly decreased（P<0.01）,while serum E₂ and INHB levels were significantly increased（P<0.05）.Compared with low-dose group,FSH was decreased and E₂ was increased in high-dose group（P<0.01）,while INHB level in medium-dose group was increased（P<0.05）.Compared with the medium-dose group,the level of E₂ in high-dose group was increased（P<0.05）.（3）Comparison of ovarian granulosa cell apoptosis rate:Compared with normal group,granulosa cell apoptosis rate in model group was significantly increased（P<0.01）;Compared with model group,granulosa cell apoptosis rate in medium and high dose groups and estrogen groups was significantly decreased（P<0.01）.Compared with the low dose group,the apoptosis rate of granulosa cells in the medium and high dose groups was decreased,and the difference was statistically significant（P<0.05）.（4）Comparison of the m RNA expression levels of Bax,Bcl-2,ROS and SOD-2:Compared with normal group,the m RNA expressions of Bcl-2 and SOD-2 in ovarian tissue of rats in model group were significantly decreased（P<0.05）,while the m RNA expressions of Bax and ROS were significantly increased（P<0.01）.Compared with model group,the m RNA expression levels of Bax and ROS in low-dose,medium-dose and high-dose groups and estrogen groups were significantly decreased（P<0.05）,while the m RNA expression levels of Bcl-2 in high-dose and estrogen groups were significantly increased（P<0.05）.The m RNA expression level of SOD-2 in estrogen group was significantly increased（P<0.05）,and the m RNA expression level of SOD-2 in low,medium and high dose groups had an increasing trend,but the difference was not statistically significant（P>0.05）.Compared with the medium dose group,the m RNA expression level of Bax in the high dose group was significantly decreased（P<0.05）.（5）Comparison of ROS content in ovarian tissue:Compared with normal group,ROS content in ovarian tissue of rats in model group was significantly increased（P<0.01）;Compared with model group,ROS content in ovarian tissue of rats in estrogen group was significantly decreased（P<0.01）,while that in other administration groups had a decreasing trend,but the difference was not statistically significant（P>0.05）.（6）Comparison of the protein expression levels of Bax,Bcl-2 and SOD-2:Compared with normal group,the protein expression levels of Bcl-2 and SOD-2 in ovarian tissue of rats in model group were significantly decreased（P<0.01）,while the protein expression level of Bax was significantly increased（P<0.05）;Compared with model group,the protein expression level of Bcl-2 in medium-dose and high-dose groups and estrogen groups was significantly increased（P<0.05）,while the expression of Bax in high-dose groups and estrogen groups was decreased and the expression of SOD-2 was increased（P<0.05）.Compared with low dose group,the expression of SOD-2 in high dose group was increased（P<0.05）.Conclusions1.Excessive oxidative stress promotes ovarian granulosa cell apoptosis,which may be one of the biological bases of liver depression and kidney deficiency syndrome of premature ovarian failure.2.Soothing the liver and tonifying the kidney can reduce the apoptosis of granulosa cells by inhibiting oxidative stress,thus regulating the endocrine function of the ovary,repairing the damaged ovary and improving the premature ovarian failure.