| Diminished ovarian function is a common problem in the field of gynecological endocrinology and reproduction.The etiology of diminished ovarian function is complex,current study has not yet explicit its pathogenesis,and western medicine is still lack of very effective treatment measures.The aggravation of it can lead to failure of the ovarian function.In recent years,Traditional Chinese Medicine(TCM)has been widely used in clinical treatment and experimental research of this problerm.But it also faces many challenges.For example,lack of Chinese patent medicine,the mechanism of TCM is not clear.Yangyin Shugan(YYSG)granule is an effective formula based on clinical practice of professor Wang Xiaoyun over 30 years.The prescription is clinically effective used to improve ovarian function,and there are no reports of adverse reactions and side effects.This study will further explore the efficacy,mechanism and safety evaluation of the drug,provide experimental basis for the treatment of diminished ovarian function,and provide scientific basis for the development of new drugs.Objective 1.To study the effect of YYSG granule on ovarian function in rats with diminished ovarian function caused by VCD,and to further explore its mechanism 2.To study the effect of YYSG granule in different dosage,and to evaluate the safety of the drug.Methods 1.Study on the efficacy and mechanism of YYSG granule on diminished ovarian function model rats SD female rats with estrous cycle rules were selected,and 10 rats of them were taken as blank group.In addition to the blank group,the other SD female rats were intraperitoneally injected with 160mg/kg VCD everyday,and continuously for 15 days.After the rat model of diminished ovarian function was established,56 rats randomly divided into 5 groups: model group,low dose of YYSG group,middle dose of YYSG group,high dose of YYSG group and Kuntai capsule group,each group had 11 rats(model group had 12 rats).All of rats were given daily gavage and continued for 4 weeks.Observe and measured the general condition,body weight changes,estrous cycle,ovarian ovarian index,ovarian histology,follicle count,the level of serum sex hormones(FSH,AMH and E2),the level of oxidant-related substances(SOD,GSH-PX and MDA),expression of PI3 K,Akt and Fox O3 a protein and apoptosis factor(Bcl-2 protein,Bax protein,Caspase-3 protein)in ovarian tissue.The data were statistically analyzed using SPSS 22.0 statistical software.2.Study on safety evaluation of YYSG granules 160 female SD rats,5~6 weeks old,weighing 140~160 g.After 1 week of adaptive feeding,the rats were randomly divided into: blank group,low dose of YYSG group,medium dose of YYSG group and high dose of YYSG group.Distilled water,0.26 g extract/m L,0.51 g extract/m L,and 1.02 g extract/m L were orally administered orally once a day(at 15 m L/kg)and continued for 3 months(13 weeks).Observe the effects of YYSG granules on the general condition,hematology,liver and kidney function of rats.The data were statistically analyzed using SPSS 22.0 statistical software.Results 1.Study on the efficacy and mechanism of YYSG granule on diminished ovarian function model rats(1)Effects of YYSG granule on general condition and estrous cycle(1)The general situation of rats Compared with the blank group,the rats in the model group had disordered abdominal hair,weakened activities,reduced diet,obvious abdominal distension,and diarrhea.Compared with the model group,the general condition of the rats in YYSG groups gradually improved,abdominal distension and diarrhea a appeared to have decreased;the general conditions(activity,diet,etc.)of rats in Kuntai capsule group also gradually improved,but there was no reduction in abdominal distension and diarrhea.(2)Rats' weight After 4 weeks of administration,the body weight of every group showed an increasing trend compared with 2 weeks of administration.Compared with blank group,the weight gain of model rats was slow,the difference between the two groups was statistically significant(P<0.01).Compared with model group,the weight of rats in every administration group increased,but the differences were not statistically significant(P?0.05);Compared with the control group,the effect YYSG groups were similar to that of Kuntai capsule group,and the differences were not statistically significant(P?0.05).No difference was found between the middle dose group with the low dose group and high dose group(P?0.05).(3)The estrous cycle of rats Compared with the blank group,the estrus cycle of model group prolonged,and the difference was statistically significant(P<0.01).Compared with the model group,the estrus cycle of the middle dose group,high dose group and Kuntai capsule group were significantly improved,and the differences were statistically significant(P<0.05).Compared with the control group,the middle and high dose groups were similar to that of Kuntai capsule group,with no statistically significant difference(P?0.05);The middle dose group was superior to the low dose group,and the difference was statistically significant(P<0.05).(2)Effect of YYSG granule on ovarian weight and ovarian index(1)Ovarian weight Compared with the blank group,the bilateral ovarian weights of the model group rats were significantly reduced,and the differences were statistically significant(P<0.01).Compared with the model group,the ovary weights of rats in YYSG groups were significantly increased(P<0.05,P<0.01).Compared with the control group,the high dose group were better than Kuntai capsule group,and the differences were statistically significant(P<0.05).No significant difference was found in the middle dose group compared with the low and high dose groups(P?0.05).(2)Ovarian index Compared with the blank group,the ovarian index of the model group rats decreased significantly(P<0.01).Compared with the model group,the ovarian index was increased in YYSG groups and Kuntai capsule group,and the differences were statistically significant(P<0.05,P<0.01).Compared with the control group,the high dose group was better than the Kuntai capsule group(P<0.01).There was no significant difference between the middle dose group with the low and high dose groups(P?0.05).(3)Effect of YYSG granule on ovarian morphology and follicular count(1)Ovarian morphology Compared with the blank group,the ovarian structure of the model group was disordered,with hyperplasia and fibrosis in the cortex,loosely arranged granulosa cells,decreased number of developed and mature follicles,and increased numbers of atresia follicles,inflammatory infiltration,and atrophy of the medulla.Compared with the model group,the ovariectomized rats showed less hyperplasia in all administration groups,normal forms of follicles were seen,the number of primordial follicles and primary follicles increased,and the number of atresia follicles decreased,there was no significant atrophy of the medulla.(2)Follicular count Compared with the blank group,the number of primordial follicles and primary follicles in the model group decreased significantly(P<0.05,P<0.01);the number of secondary follicles and mature follicles increased,but there was no statistical difference(P?0.05);The number of atresia follicles increased,and the difference was statistically significant(P<0.01).Compared with the model group,the number of developed and mature follicles tend to increase in all administration groups,but the differences were not statistically significant(P?0.05).In terms of reducing the number of ataxia follicles,the number of low dose group,middle dose group and Climen group was reduced compared with the model group(P<0.05);Compared with the control group,the efficacy of the low group and middle dose group was comparable to that of Climen group,there was no statistical difference(P?0.05);the efficacy of the low dose group was better than Kuntai capsule group,and the difference was statistically significant(P<0.05).(4)Effect of YYSG granule on serum sex hormone level Compared with the blank group,in the model group,the serum FSH levels increased,E2 and AMH levels decreased,the differences were statistically significant(P<0.05).Compared with the model group,in drug administration groups,the serum FSH levels showed a decreasing trend,and the serum E2 and AMH levels showed increasing trends.The differences between the high-dose group with Kuntai capsule group and Climen group were statistically significant(P<0.05).There was no significant difference between the high dose group and the Kuntai capsule group(P?0.05).(5)Effect of YYSG granule on oxidant-related substances Compared with the blank group,the serum GSH-PX and SOD activity in the model group were decreased and the MDA level was increased,and the differences were statistically significant(P<0.05).Compared with the model group,theactivity of serum GSH-PX and SOD in drug intervention groups showed a rising trend and the MDA level showed a downward trend,the differences of high dose group and Kuntai capsule group were statistically significant(P<0.05).There was no significant difference between the high-dose group with Kuntai capsule group(P?0.05).(6)Effect of YYSG granule on PI3K/Akt/Fox O3 a pathway associated proteins in ovarian tissue(1)The expression of PI3 K protein in ovarian tissue Compared with the blank group,the expression level of PI3 K protein in the model group was significantly down-regulated(P<0.01).Compared with the model group,PI3 K protein expression was significantly increased after intervention of m iddle dose,high dose and Kuntai capsules,the differences were statistically significant(P<0.01).Compared with the control group,the efficacy of the middle dose group and high dose group was similar to that of the Kuntai capsule group,with no statistically significant difference(P?0.05).There was no significant difference between the middle dose group with the low and high dose groups(P?0.05).(2)The expression of Akt protein in ovarian tissue Compared with the blank group,the expression level of Akt protein in the model group was significantly down-regulated(P<0.01).Compared with the model group,the expression level of Akt protein was significantly up-regulated in all groups,and the differences were statistically significant(P<0.01).Compared with the control group,the Kuntai capsule group was superior to the low-dose group,and the difference was statistically significant(P<0.01).YYSG groups showed an increasing effect,the difference was statistically significant(P<0.01,P<0.01).(3)The expression of Fox O3 a protein in ovarian tissue Using immunohistochemical method,compared with the blank group,the expression level of Fox O3 a protein in the model group was significantly up-regulated(P<0.01);the IOD of Fox O3 a protein in the rats of all administration group were significantly decreased,and the difference were statistical significance(P<0.01).Compared with the control group,the middle and high dose groups were better than the Kuntai capsule group,the difference was statistically significant(P<0.05,P<0.01).There was no significant difference between the middle dose group and the low and high dose groups(P?0.05). Western blot analysis showed that the expression of Fox O3 a protein in the model group was significantly higher than that in the control group(P<0.01).The expression of Fox O3 a protein was significantly reduced in the rats of each treatment group(P<0.01).Compared with the control group,the middle and high dose group was better than Kuntai capsule group,and the difference was statistically significant(P<0.01).YYSG groups showed an increasing effect,the differences were statistically significant(P<0.01,P<0.01).(7)Effect of YYSG granule on the expression level of apoptosis-related protein in ovarian tissue(1)The expression level of Bcl-2 protein in ovarian tissue Compared with the blank group,the expression of Bcl-2 protein in the model group was significantly decreased,and the difference was statistically significant(P<0.01).Compared with the model group,the expressions of Bcl-2 protein in drug intervention groups were significantly increased,and the differences were statistically significant(P<0.01).Compared with the control group,the middle dose group was better than Kuntai capsule group,and the difference was statistically significant(P<0.01).The middle dose group was superior to the low dose group and high dose group,and the differences were statistically significant(P<0.01).(2)The expression level of Bax protein in ovarian tissue Compared with the blank group,the expression of Bax protein in the model group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the model group,the expression of Bax protein in drug intervention groups were significantly reduced,and the differences were statistically significant(P<0.01).Compared with the control group,the high dose group was superior to Kuntai capsule group,the difference was statistically significant(P<0.01).YYSG groups showed an increasing effect,the difference were statistically significant(P<0.01).(3)The expression level of Caspase-3 protein in ovarian tissue Compared with the blank group,Caspase-3 protein expression in model group rats was significantly increased,the difference was statistically significant(P<0.01).Compared with the model group,the expressions of Caspase-3 protein in drug intervention groups were significantly reduced,and the differences were statistically significant(P<0.01).Compared with the control group,the midle and high dose group was better than Kuntai capsule group,the differences were statistically significant(P<0.05,P<0.01).YYSG groups showed an increasing effect,the difference was statistically significant(P<0.05).2.Experimental study on safety evaluation of YYSG granule(1)Effects of YYSG granule on general condition of rats During the experiment,none of the rats in each group died and there was no obvious toxic reaction.Compared with the blank group,the appearance,behavior,diet,and excretion of the rats in the low-,middle-,and high-dose groups were normal.(2)Effects of YYSG granule on hematology index Compared with the blank group,the count of WBC,Neu,and PLT in all groups were normal,there was no significant difference between the low-,middle-,and high-dose groups with blank group(P?0.05);The count of RBC and HGB content in YYSG group decreased,and the difference was statistically significant(P<0.01).(3)Effects of YYSG granule on index of liver function and kidney function In terms of liver function(levels of serum ALT and AST),there was no significant difference between the low-,midle-,and high-dose groups with blank group(P?0.05).In terms of renal function(serum BUN,CRE),there was no significant difference between the low-,midle-,and high-dose groups with blank group(P?0.05).Conclusion 1.The model was successfully established by intraperitoneal injection of 160 mg·kg-1 VCD for 15 days.2.YYSG granule could improve the general condition of rats with diminished ovarian function,especially in reducing abdominal distention.3.YYSG granule could promote the recovery of estrous cycle in diminished ovarian function model rats.4.YYSG granule could increase both the ovarian weight and ovarian index in diminished ovarian function model rats,especially in the midle and high dose.5.YYSG granule could improve the ovarian tissue structure in diminished ovarian function model rats,increase the numbers of developed follicles and mature follicles;YYSG granule could significantly reduce the numbers of atresia follicles.6.YYSG granule could regulate the level of sex hormones(AMH,FSH,and E2)in diminished ovarian function model rats,reduce the level of serum FSH,and increase the levels of serum AMH,E2,especially in the high dose.7.YYSG granule could exert antioxidant effects,improve serum GSH-PX,SOD activity,and reduce MDA levels,especially in the high dose.8.YYSG granule could regulate the expression of PI3K/AKT/Fox O3 a pathway related proteins in ovarian tissues,increase the expression of PI3 K and A kt proteins,and decrease the expression of Fox O3 a protein.9.YYSG granule could regulate the expression of apoptosis related proteins in ovarian tissues and inhibit apoptosis,increase the expression of Bcl-2 protein,and decrease the expression of Bax,Caspase-3 protein.10.Rats in the safety evaluation experiment were generally in good condition,without death and toxicity,no obvious hematological changes and damage t o liver and kidney function,and preliminary showed the safety of YYSG granule.11.YYSG granule could improve the ovarian function in diminished ovarian function model rats.The mechanism may be through regulating the signal pathway of PI3K/AKT/Fox O3 a in ovarian tissues and inhibiting apoptosis,and the drug was safe.This study provided experimental basis for the promotion and application of YYSG granule. |
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