An Experimental Study Of Zuogui Pill Combined With Cetrorelix To Balance Follicular Oxidative Stress Response By SIRT1/P53/P21 Pathway To Improve The Decline Of Ovarian Reserve Caused By Cyclophosphamide

Objective:To observe the effect of tonify kidney and nourish Yin recipe Zuogui Pill combined with gonadotropin-releasing hormone antagonist cetrorelix on balancing the oxidative stress response in the follicle microenvironment,and to study the combination of Chinese and Western methods to improve the decline of ovarian reserve caused by cyclophosphamide Mechanisms.Method:Female ICR mice were divided into blank group?group A?,model group?group B?,cetrorelix group?group C?,Zuoguiwan group?group D?,and Chinese and western combination group?group E?.The first day is called d1,and the last day of the experiment is d38.cetrorelix was given to groups C and E in d1-d5,and the other three groups were given saline.CTX was given to groups B,C,D,and E in d6-d10,and saline was given to group A;Zuogui pill medicine was given to groups D and E in d11-d38,the other three groups were given water.The levels of FSHR,E2,LH,AMH,GDF-9,BDNF,LIF,NO,NOS and ROS in ovarian homogenate were detected by SA ELISA,FSH and FSHR levels in peripheral blood were detected,and the expressions of p53,p21 and SIRT1 proteins in ovary were detected by immunohistochemistry and western blot methods.Result:1?body weight:The body weight of the blank group increased steadily within38 days of the experiment cycle,the other groups continued to drop rapidly after d6,and the animals showed phenomena such as depilation,malaise and thin stools.Starting from d11,the body weight of cetrorelix group,zuogui pill group and the combination group began to rise again.By d38,the body weight of the zuogui pill group almost recovered to the same level as the blank group,and the body weight of the cetrorelix group and the combination group also recovered to a certain extent.The model group had been losing weight rapidly and animals were dying.2?Estrus period and interestrus period:compared with the blank group,the number of days of estrus period in the model group decreased significantly?P<0.01?.In contrast,the interestrus period was longer than the blank period?P<0.01?,and the data of both groups were statistically significant.Cycle length:Compared with the blank group,the estrous cycle length of the model group was significantly longer?P<0.01?,and the cycle length of the combination group was similar to the blank group,which was statistically significant compared with the model group?P<0.01?..3?HE staining results of ovarian sections showed that:primordial follicles:compared with the blank group,the number of primordial follicles in the model group was significantly reduced?P<0.01?.Compared with the model group,the number of primary follicles in the three treatment groups was higher,and the differences were statistically significant?P<0.01?.Growing follicles:compared with the blank group,the number of growing follicles in the model group was significantly reduced.The number of growing follicles in the combination group was close to the level of the blank group,and the difference was statistically significant?P<0.05?.Atretic follicles:compared with the blank group,the number of original follicles in the model group increased significantly,but the difference was not statistically significant?P>0.05?.Compared with the model group,the atretic follicles of the three treatment groups decreased,but the difference was not statistically significant?P>0.05?.4?Immunohistochemical results showed that:SIRT1:compared with the blank group,SIRT1 protein's expression was significantly reduced in the model group.Compared with the model group,the expression of SIRT1 protein increased in the three treatment groups,of which the zuogui pill group and the combination group were more obvious.P53:P53 protein's expression increased significantly in the model group compared with the blank group.Compared with the model group,the expression of P53 protein in the cetrorelix group and zuogui pill group decreased,and the expression of P53 protein in the combination group significantly decreased,which was close to the level of the blank group.P21:compared with the blank group,P21protein's expression was significantly increased in the model group.Compared with the model group,the P21 protein expression was decreased in the cetrorelix group and the zuogui pill group,and the P21 protein expression was significantly decreased in the combination group,which was close to the level of the blank group Detection of serum basal hormone levels in each group5?ELISA method detected serum and ovarian hormones and AMH levels showed:Serum FSH:model group serum FSH level was significantly higher than the blank group,the difference between the groups was statistically significant?P<0.05?.Serum FSH levels in the cetrorelix group,Zuogui pill group,and combination group decreased,of which the Zuogui pill group and the combination group decreased significantly,with a statistically significant difference compared with the model group?P<0.01?;Serum E₂:The serum E₂concentration of the model group and the blank group decreased,but the difference did not reach statistical significance?P<0.05?.The E₂concentration of the three treatment groups increased significantly compared with the model group?P<0.01?.Ovarian LH:Compared with the blank group,the LH concentration in the ovary of the model group increased?P<0.01?,and the LH level of the three treatment groups decreased?P<0.01?.Ovarian E₂:Compared with the blank group,the E₂concentration of the model group decreased?P<0.01?.The E₂concentration in the zuogui pill group and the combination group increased,but the difference was not statistically significant compared with the model group?P>0.05?.Ovarian AMH:Compared with the blank group,the AMH concentration in the ovaries of the model group decreased?P<0.01?.After treatment,the AMH concentration in the Zuogui pill group and the combination group returned to the level of the blank group.The difference is significant?P<0.01?.Ovarian FSHR:Compared with the blank group,the FSHR concentration of the model group decreased?P<0.05?,and the FSHR concentration of the three treatment groups increased after treatment.The difference between the combination group and the model group was the most obvious?P<0.01?.6?ELISA for the detection of oxidative stress related factors in each group showed that,compared with the blank group,the NOS concentration in the model group increased?P<0.05?,and after treatment,the concentration in the zuogui pill group and the combination group decreased as compared with the model group,with statistically significant differences?P<0.05?.Compared with the blank group,the concentration of NO in the model group increased?P<0.01?,and the concentration of NO in the three treatment groups decreased?P<0.01?.Compared with the blank group,the ROS concentration of the model group increased significantly?P<0.01?,and the ROS concentration of the three treatment groups all decreased.The concentration of the combination group was similar to the blank group,and the difference was significant compared with the model group?P<0.01?.Compared with the blank group,ATP concentration in the model group decreased significantly?P<0.05?,ATP concentration in the three treatment groups increased,among which in the Combination group was similar to the blank group,and the difference between the Zuogui pill group and the model group was significant?P<0.01?.7?The growth and development related factors of each group were detected by sa method:compared with the blank group,the DBNF and GDF-9 concentrations of the model group were significantly decreased?P<0.01?.After treatment,the concentration of BDNF in the three treatment groups increased,and the combination group returned to the level of the blank group.Compared with the model group,the difference was statistically significant?P<0.01?.In addition,the concentration of GDF-9 in the three treatment groups all increased to the level of the blank group,and the difference was statistically significant?P<0.01?.Compared with the blank group,LIF level in the model group decreased,and LIF concentration in the three treatment groups increased after treatment,but the difference was not statistically significant.Conclusion:1.The chemotherapeutic drug cyclophosphamide can induce excessive oxidative stress in the ovaries,resulting in ovarian function damage and decreased ovarian reserve.In this animal experiment,CTX 50mg/kg was used for 5 consecutive days,which met the diagnostic criteria of an increase in serum hormone FSH and a decrease in E2 in the ovary.2.The SIRT1/P53/P21 pathway is involved in the occurrence of CTX-induced DOR,and its mechanism may be related to the inhibition of local ovarian oxidative stress.3.The gonadotropin-releasing hormone antagonist cetrorelix combined with tonifying kidney and tonifying yin Recipe Zuogui Pill can improve the oxidative stress response in the follicle microenvironment by regulating the SIRT1/P53/P21pathway and reduce the production of peroxidation products to a certain extent The effect of repairing DOR caused by CTX is better than that of Cetrorelix or Zuogui pills alone.But its exact mechanism needs further study.