| Objective:We duplicated the androgen induced sterile rats(ASR), made the normal SD rat and ASR as object of investigation, at the same time, we interfered with medicine. And we aproached the action of hemin on HO/bilirubin and ovarian function, the action on HO/bilirubin system in ASR,as well as the effect of Bushentiaojing Recipe on them; We studied the anti-oxidantive action on HO/bilirubin and ovarian function in normal rats,the action on anti-oxidation of HO/bilirubin system in ASR,as well as the effect of Bushentiaojing Recipe on them;We investigated anti-apoptosis action of HO/bilirubin system on normal and anovulatory ovary , as well as anovulatory ovary administrated by Bushentiaojing Recipe;In addition,we observed the action of Bushentiaojing Recipe on them。Methods Part 1 : Study of HO/bilirubin system and ovarian function in normal rat Experiment 1 Study of HO-1 activity and serum bilirubin in normal rat 20 healthy female rats of 3-4-month age were divided into 2 groups at random , namely Normal group and Hemin group. Hemin group subcutaneously injected hemin in nape,30μm/kg in dosage;Normal group subcutaneously injected normal saline in nape for successive 7 days. While making vagina cells smear,sacrificed the animal at the postoestrus and compared the level of HO-1 activity and serum total bilirubin,indirect bilirubin and direct bilirubin of two groups.. Experiment 2: Relation of HO/bilirubin and oxidation, anti-oxidation in normal rat The group distribution,drug-administration and animal sacrifice were exactly the same as above. Observed and compared the level of MDA and T-AOC in serum(plasum)and ovary tissue. Experiment 3: Action of hemin on the apoptosis expression of ovary in normal rat The group distribution,drug-administration and animal sacrifice were exactly the same as above. Observed and compared the expression of granular cell apoptosis and caspase-3. Experiment 4: Study of HO/bilirubin and function of ovary in normal rat The group distribution,drug-administration and animal sacrifice were exactly the same as above. Observed and compared the level of sexual hormone,pathomorphology and index number of ovary. Part 2: The study of HO/bilirubin in ASR Experiment 1: Study of HO-1 activity and serum bilirubin in ASR The anovulatory model was established by subcutaneous injection of testoserone propionate to SD female rats of 9-day age. Normal rats injected isodose neutral tea oil as control. When the rats were 80-day age,we began to make the smear of vaginal epithelial cell for 2 successive sexual cycle(4-5 days per sexual cycle), euthanized at the preoestrus. Vaginal epithelial cell showed continuously cutinization , which hinting successful model of anovulatory. They all lavaged with distilled water. While making vagina cells smear, euthanized the animals at the preoestrus, and compared the level of HO-1 activity and serum total bilirubin,indirect bilirubin and direct bilirubin.. Experiment 2: Relation of HO/bilirubin and ovarian oxidation ,anti-oxidation in ASR The group distribution,drug-administration and animal sacrifice were exactly the same as above. Observed and compared the level of MDA and T-AOC in serum(plasum)and ovary tissue homogenate. Experiment 3: Study of HO/bilirubin and the expression of granular apoptosis of ovary in ASR The group distribution,drug-administration and animal sacrifice were exactly the same as above. Observed and compared the expression of granular cell apoptosis and caspase-3. The high clarity pathological system was used to measure positive cell mean integral light density byqualitative-semiquantitative analysis . Experiment 4: Study of HO/bilirubin and ovarian function in ASR The group distribution,drug-administration and animal sacrifice were exactly the same as above. Observed and compared the level of sexual hormone, pathomorphology and index number of ovary. Part 3: Effect of Bushen tiaojing Recipe on ovarian HO/bilirubin system in ASR Experiment 1: Effect of Bushentiaojing Recipe on function of ovary in ASR The anovulatory model was established by subcutaneous injection of testoserone propionate to SD female rats of 9-day age. Normal rats injected isodose neutral tea oil as control. When the rats were 80-day age,we began to make the smear of vaginal epithelial cell for 2 successive sexual cycle(4-5 days per sexual cycle), euthanized at the preoestrus. Vaginal epithelial cell showed continuously cutinization , which hinting successful model of anovulatory .40 female ASR were randomly divided into the model group,the model group treated with high dose(53g·kg-1·d-1,correspondent to 20 times of clinical dosage) and low dose of herbs(27g·kg-1·d-1,correspondent to 10 times of clinical dosage),the model group treated with Wujibaifeng Oral Liquid(1ml·kg-1·d-1,correspondent to 20 times of clinical dosage),10 rats of the same age were made as normal control lavaged with distilled water . While making vagina cells smear,euthanized the animals at the preoestrus. Observed and compared the level of sexual hormone, pathomorphology and index number of ovary. Experiment 2: Effect of Bushentiaojing Recipe on ovarian oxidation and anti-oxidation in ASR The group distribution,drug-administration and animal sacrifice were exactly the same as above. Observed and compared the level of MDA and T-AOC in serum(plasum)and ovary tissue homogenate. Experiment 3: Effect of Bushen tiaojing Recipe on the expression of granular cell apoptosis and caspase-3 of ovarian in ASRThe group distribution,drug-administration and animal sacrifice were exactly the same as above. Observed and compared the expression of granular cell apoptosis and caspase-3.The high clarity pathological system was used to measure positive cell mean integral light density by qualitative-semiquantitative analysis . Experiment 4: Effect of Bushentiaojing Recipe on granular apoptosis of ovary in ASR The group distribution,drug-administration and animal sacrifice were exactly the same as above. and compared the expressionl of granular apoptosis and caspase-3 protein. All the data mentioned above were analyzed with SPSS 11.5 statistical software, Independent Sample T-test. multi-mean applied with analysis of variance(ANOVA),comparation between two groups with LSD. Correlation analysis with Pearson test. Results Part 1 : Study of HO/bilirubin system and ovarian function in normal rat Experiment 1 Study of HO-1 activity and serum bilirubin in normal rat The average value of HO-1 activity of hemin group and normal group was 7.46±0.83,6.55±0.58 respectively,and former were significantly higher than the latter in HO–1 activity(P<0.05). The average value of serum Tbil of hemin group and normal group was 6.80±0.25,6.03±0.20 respectively,and the former were significantly higher than the latter in HO–1(P<0.01);The average value of serum Ibil of hemin group and normal group was 4.86±0.38,4.31±0.20 respectively,The average value of serum Dbil of hemin group and normal group was 1.94±0.29,1.72±0.11 respectively,compared with normal group,there was significant difference in the level of Ibil and Dbil between two groups(P<0.05). Experiment 2: Relation between HO/bilirubin and ovarian oxidation, anti-oxidation in normal rat The average value of plasum MDA of hemin group and normal group was 0.056±0.005 , 0.062±0.005 respectively , and the former weresignificantly lower than the latter in HO–1(P<0.01);The average value of ovarian tissue homogenate MDA of hemin group and normal group was 0.150±0.176 ,0.229±0.147 respectively,and former were significantly lower than the latter in MDA(P<0.01). The average value of serum T-AOC of hemin group and normal group was 0.082±0.016,0.065±.012 respectively,and former were significantly higher than the latter in serum T-AOC(P<0.05);The average value of ovarian tissue homogenate T-AOC of hemin group and normal group was 3.672±1.558,4.275±1.134 respectively,and former were significantly higher than the latter in ovary T-AOC(P<0.01). Analysis of correlation between bilirubin and MDA,T-AOC in each groups The experimental result indicated that there was negative correlation between Ibil and MDA,r=-0.562,P<0.05; there was positive correlation between Ibil and T-AOC,r=0.531,P<0.05. Experiment 3: Effect of hemin on expression of granular apoptosis of ovary in normal rat (1)The apoptosis in ovary There were apoptosis in ovary tissure to some degree. The expression of theca cell apoptosis was most significant, while the weakest in corpus lutea, granular cell, stroma and the interstitial cells were in the middle of them. By induced of hemin , the expression of apoptosis significantly decreased in copus lutea ,theca and granular cells (P<0.01). The mean integral light density of the expression of granular cell apoptosis cell of normal group and hemin group was 107.23±2.79,130.10±3.50 respectively,and former were significantly higher than that of the latter(P<0.05). The mean integral light density of the apoptosis expression of copus lutea cell of normal group and hemin group was 128.35±4.59,143.58±5.21 respectively,and the latter were significantly weaker than that of the former(P<0.05). The mean integral light density of the apoptosis expression of theca cell of normal group and hemin group was 87.75±5.53,102.83±7.85 respectively,and the latter were significantly weaker than that of the former(P<0.05). (2)The protein expression of caspase-3 in ovary The mean integral light density of the apoptosis expression of granular cell of normal group and hemin group was 117.75±5.53,130.90±10.00 respectively,and the latter were significantly weaker than the forme(rP<0.05). The mean integral light density of the caspase-3 expression of copus lutea cell of normal group and hemin group was112.32 ±10.21 ,150.4 ±7.33 respectively,and the latter were significantly weaker than that of the former(P<0.01). The mean integral light density of the caspase-3 expression of theca cell of normal group and hemin group was 98.18±3.58,113.98±15.96 respectively,and the latter were significantly weaker than that of the former(P<0.05). Experiment 4: Relation between HO/bilirubin and function of ovary in normal rat In two groups,the construction of each layer in follicular was normal,the typical corpus lutea was seen to form. But the vessels in corpus lutea of hemin group was much obviously. The average value of serum E2 of hemin group and normal group was 6.986±3.074,3.871±2.839 respectively,and the former were significantly higher than the latter(P<0.05); The average value of serum P of hemin group and normal group was 6.801±1.474,3.969±2.066 respectively, and former were significantly higher than the latte(rP<0.01);The average value of serum T of hemin group and normal group was 3.086±1.163 , 2.444±1.784 respectively,there was no significant difference in the level of T(P>0.05). Analysis of correlation between bilirubin and sexual hormone in each groups: The experimental result indicated that there was positive correlation between P and Tbil, Ibil, Dbil,r=0.494,0.777,0.619 respectively, P<0.01, 0.05, 0.01 respectively; there was positive correlation between Ibil and E,r=0.553,P<0.05. Part 2: The study of HO/bilirubin system in ASRExperiment 1: The study of HO-1 activity and serum bilirubin in ASR The average value of HO-1 activity of normal group,model group was 6.96±1.35, 6.19±0.78 respectively. Compared with model group, HO-1 activity of normal group was decreased(P<0.05). The average value of serum Tbil of normal group, model group 6.47±1.18,5.79±0.36 was respectively. Compared with model group, serum Tbil of normal group was increased(P<0.05). The average value of serum Ibil of normal group, model group was 4.45±0.55, 4.08±0.27 respectively . Compared with model group, serum Ibil of normal group was increased(P<0.05). The average value of serum Dbil of normal group, model group was 2.02±1.16,1.71±0.42 respectively. Compared with model group, serum Dbil of normal group have no significantly difference(P>0.05). Experiment 2: Relation between HO/bilirubin and ovarian oxidation, anti-oxidation in ASR The average value of plasum MDA of normal group, model group was 0.06±0.01,0.07±0.01 respectively . Compared with normal group, plasum MDA of normal group was significantly increased(P<0.05). The average value of ovarian MDA of each group was 0.08±0.01,0.09±0.004 respectively,Compared with normal group, MDA of model group was significantly increased(P<0.01). The average value of serum T-AOC of each group was 0.075±0.027,0.066±0.005 respectively ; Compared with normal group, that of model group was significantly decreased(P<0.05). The average value of ovarian tissue T-AOC of each group was 0.253±0.010,0.225±0.015,Compared with normal group, that of model group was significantly decreased(P<0.05). Analysis of correlation between bilirubin and T-AOC in each groups The experimental result indicated that there was negative correlation between Tbil and MDA,r=-0.562,P<0.05; there was negative correlation between Dbil and MDA,r=-0.508,P<0.05. there was positive correlationbetween Ibil and T-AOC,r=0.655,P<0.01. Experiment 3: Relation between HO/bilirubin and the expression of granular apoptosis of ovary in ASR The mean integral light density of the expression of granular cell apoptosis of normal group,model group was 165.7±6.2,116.9±12.0 respectively .Compared with normal group,the expression of granular cell apoptosis of model group was significantly increased(P<0.01). The mean integral light density of the expression of granular cell caspase-3 of normal group,model group was 113.5±5.6,66.3±4.9 respectively . Compared with normal group,the expression of granular cell caspase-3 of model group was significantly increased(P<0.01). Experiment 4: Study of HO/bilirubin and ovarian function in ASR The animal's general condition of each group:The rats of model was fat, lazy and unsensitive. The normal rats grew well, their color pattern were white and luster, their spirit and reaction is healthy, food,drink ,body weight, and urine and stool were normal. The average value of body weight of normal group,model group was 233.15±5.62, 274.32±7.83 respectively;Compared with normal group,that of model group was significantly increased(P<0.01).The average value of ovarian index of normal group,model group was 0.037±0.002,0.014±0.003 respectively;Compared with normal group , that of model group was significantly decreased(P<0.01). The average value of serum E2 of normal group,model group was 5.67±4.23,10.52±4.45 respectively;The average value of serum T was 1.11±0.27,3.39±0.19 respectively;The average value of serum P was 5.12±1.36,5.76±1.15 respectively. Compared with model group,serum E2 and T of normal group was significantly lower(P<0.05),there were no obviously difference in P(P>0.05)。Analysis of correlation between bilirubin and Sexual hormone in each groups: There were negtive correlation between Tbil and T, r=-0.325, P<0.05. There were negtive correlation between Ibil and E2 ,T, r=-0.404, -0.527respectively, P<0.05, P<0.01. Ovarian morphological observation: Compared with the normal group,there were many developed well follicular in different grades,the construction of each layer in follicular was lampros,the typical corpus luteum was seen to form.while in the model group, there was no typical corpus luteum and the follicular developed not well. Part 3: Effect of Bushentiaojing Recipe on ovarian HO/bilirubin system in ASR Experiment 1: Effect of Bushentiaojing Recipe on function of ovary in ASR The animal 's general condition of each group:The rats of model was fat, lazy and unsensitive. The other rats grew well, their color pattern were white and luster,their spirit and reaction is healthy, food,drink ,body weight,and urine and stool were normal. The average value of body weight of normal group,model group,high-dose group,low-dose group and Wujibaifeng group was 233.15±5.62, 274.32±7.83, 226.87±4.35, 241.31±5.14, 259.43±7.52 respectively;The average value of ovarian index of normal group,model group,high-dose group,low-dose group and Wujibaifeng group was 0.037±0.002,0.014±0.003,0.040±0.008,0.028±0.006,0.017±0.002 respectively,Compared with model group,that of other groups was significantly increased(P<0.01),there were obviously difference between high-dose and low-dose group and between high-dose and Wujibaifeng group(P<0.05). Ovarian morphological observation Compared with the control group and model group,there were many developed well follicular in different grades,the construction of each layer in follicular was lampros,the typical corepus luteum was seen to form. The average value of serum E2 of normal group,model group, high-dose group,low-dose group and Wujibaifeng group 5.67±4.23,10.52±4.45,6.13±1.87,6.29±3.85,9.56±1.38 respectively;The average value of serum T was 1.11 ±0.27 ,3.39 ±0.19 ,1.11 ±0.13 ,1.21 ±0.21 ,1.60 ±0.97respectively;The average value of serum P was 5.12±1.36,5.76±1.15,5.23±0.48,5.15±1.53,5.03±0.70 respectively. Compared with model group,serum E2 and T of of three herb group was significantly lower(P<0.01),Compared with model group, that of high-dose group , low-dose group and Wuji group was significantly lower(P<0.01),there were no obviously difference in P in low-dose group and Wuji group(P>0.05),there were no obviously difference in P(P>0.05) Experiment 2:Relation between of Bushentiaojing Recipe on ovarian HO-1 activity and serum bilirubin in ASR The average value of HO-1 activity of normal group,model group,high-dose group , low-dose group and Wujibaifeng group 6.96±1.35 ,6.19±0.78,7.32±0.58,6.98±0.21,6.98±0.48 respectively. Compared with model group, HO-1 activity of low-dose group was significantly increased ( P < 0.05 ), that of high-dose group and Wujibaifeng group were significantly increased(P<0.01). The average value of serum Tbil of normal group,model group,high-dose group , low-dose group and Wujibaifeng group 6.47±1.18 ,5.79±0.36,6.44±0.42,6.18±0.54,5.93±0.29 respectively. Compared with model group, serum Tbil of normal group was increased(P<0.05),serum Tbil of high-dose group and Wujibaifeng group was significantly increased(P<0.05),that of low-dose group has no significantly difference(P>0.05). The average value of serum Ibil of normal group, model group, high-dose group, low-dose group and Wujibaifeng group 4.45±0.55, 4.08±0.27,4.53±0.19,4.31±0.26,4.69±0.32 respectively . Compared with model group, serum Ibil of normal group was increased(P<0.05),serum Tbil of high-dose group and Wujibaifeng group was significantly increased (P<0.01),that of low-dose group was significantly increased (P<0.05),while there were no significant difference in the three drug groups(P >0.05). The average value of serum Dbil of normal group, model group, high-dose group, low-dose group and Wujibaifeng group 2.02±1.16,1.71±0.42,1.92±0.45,1.87±0.59,0.26±0.10 respectively. Compared with model group, serum Dbil of other groups have no significantly differece(P>0.05). Experiment 3: Relation between of Bushentiaojing Recipe on ovarian oxidative damage and anti-oxidation in ASR The average value of plasum MDA of normal group,model group, high-dose group,low-dose group and Wujibaifeng group was 0.06±0.01,0.07±0.01,0.05±0.02,0.057±0.016,0.06±0.001 respectively .Compared with normal group,plasum MDA of normal group was significantly increased(P<0.05);Compared with model group,plasum MDA of high-dose group and low-dose group was significantly decreased(P<0.05),but that of Wujibaifeng group has no significant difference. The average value of ovarian tissue MDA of each group was 0.08±0.01, 0.09±0.004, 0.08±0.004, 0.08±0.008, 0.02±0.02 respectively,Compared with model group, MDA of normal group was significantly decreased (P<0.01), that of high-dose and low-dose group was significantly decreased(P<0.01,0.05), Compared with model group, that of Wujibaifeng group was extremely decreased(P<0.01). The average value of serum T-AOC of each group was 0.075±0.027, 0.066±0.005,0.084±0.012,0.076±0.007,0.082±0.002 respectively;Compared with model group, that of of high-dose and low-dose group was significantly increased(P<0.05), while there were no significant difference in high-dose and low-dose group(P >0.05). The average value of ovarian tissue homogenate T-AOC of each group was 0.253±0.010,0.225±0.015,0.254±0.010,0.248±0.020,0.25±0.444 respectively;Compared with model group,that of high-dose, low-dose group and Wuji group was significantly increased(P<0.01), while there were no significant difference in high-dose and low-dose group(P >0.05). Experiment 4: Effect of Bushentiaojing Recipe on the expression of granular apoptosis of ovary in ASR The mean integral light density of the expression of granular cellapoptosis of normal group, model group, high-dose group, low-dose group and Wujibaifeng group was 165.7±6.2,116.9±12.0,152.8±5.4,151.2±11.0,134.9±4.14 respectively.The result of qualitative-semiquantitative analysis showed, the sepuence of each group was as followed, model group< normal group < Wujibaifeng group < low-dose group < high-dose group.Which indicated there was more apoptosis of granular cell in ASR,Bushentiaojing Recipe can decrease more apoptosis of granular cell in ASR to different degree. The effect of Bushentiaojing Recipe were better than that of Wujibaifeng Oral liquid. The mean integral light density of the expression of granular cell caspase-3 of normal group, model group,high-dose group,low-dose group and Wujibaifeng group was 113.5±5.6,66.3±4.9,92.0±4.9,87.1±3.5,78.28±4.27 respectively.The result of qualitative-semiquantitative analysis showed,the sepuence of each group was as followed, model group |
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