| Objective:On the basis of establishing polycystic ovary syndrome(PCOS)model rats,to observe the effect of Bushen Tiaojing Decoction on the expression of Serum Homeostasis model assessment of insulin resistance(HOMA-IR),Sex hormone-binding Globulin(SHBG),Free androgen Index(FAI)and ovarian mi R-21 in model rats.To explore the pathogenesis of PCOS and the therapeutic mechanism of Bushen Tiaojing Decoction regulating androgen and insulin levels,and to provide experimental basis for clinical application of Bushen Tiaojing Decoction.Methods:After selecting female SD rats aged 7-8 weeks(weight 175-185g),feeding them adaptively and observing the estrus cycle,48 rats with normal estrus cycle were randomly divided into 12 rats in the blank group and 36 rats in the PCOS model group(blank group: PCOS model group=1:3).Letrozole solution gavage was performed in the model group by letrozole modeling method.Meanwhile,rats in the model group were fed with high-fat diet,daily drinking water was replaced by 5% glucose solution,and rats in the blank group were fed with 1%CMC solution,common feed,drinking water,and continuous gavage for 27 days to establish the PCOS rat model.After the completion of the modeling,all the rats were to smear vaginal exfoliative cell,2 rats in the blank group and 6 inthe model group were randomly selected,fasting blood glucose(FPG)was measured by blood glucose meter,testosterone(T)and fasting insulin(FINS)were measured by enzyme linked immunoassay(ELISA),and HOMA-IR value was calculated,and ovarian tissue morphology was observed to evaluate the success of the modeling.After verification of successful modeling,rats in the model group were randomly divided into 3 groups:model control group,traditional Chinese medicine group and western medicine group,with 10 rats in each group.The model control group was given 1% carboxymethyl cellulose(CMC)solution by gavage,the traditional Chinese medicine group was given Bushen Tiaojing Decoction,and the western medicine group was given metformin solution by gavage.The above three groups were fed a high-fat diet throughout the whole experiment.Replace daily drinking water with 5% glucose solution;The blank group was given by gavage of 1%CMC solution,common feed and drinking water for21 days.On the 21 st day,all the rats were deprived of water for the night,the next morning,the tail vein blood was taken to measure FPG with blood glucose meter,the rats were killed,the serum FINS,T,SHBG were measured by ELISA.The bilateral ovaries of rats were taken,one ovary was used to observe the histomorphological changes,and the other ovary was used to determine the content of mi R-21 by real-time fluorescent quantitative PCR(q RT-PCR).Results:1.Model evaluation:(1)General conditions: in the blank group,the rats were generally in good condition in terms of spirit,activity response,food intake and drinking water,etc.,while in the model group,the symptoms were obesity,fatigue,sleepiness,decreased activity,and insensitive response to external stimuli.Daily food intake,water intake and urine volume were all greater than those in the blank group.(2)Rat weight: starting from 1 week after modeling,the body weight of rats in the model group and the blank group showed significant difference(P<0.01),and the body weight of rats in the model group was much higher than that in the blank group.(3)The smear of vaginal exfoliated cells in rats indicated that the rats in the blank group all had regular estrous cycle,while the rats in the model group showed prolonged estrous period or disordered estrous cycle.(4)2 rats in the blank group and 6 rats in the model group were randomly selected(blank group: model group =1:3)to verify whether the modeling was successful.The results showed that the serum FINS and T in the model group were significantly higher than those in the blank group(P<0.05),and there was no significant difference in FPG between the model group and the blank group(P>0.05).HOMA-IR level of rats in the model group was significantly higher than that in the blank group(P<0.05).(5)Morphologic changes of rat ovarian tissue: macroscopic observation showed that the ovarian volume of the model group was larger and the ovary length diameter was longer than that of the blank group.The ovaries of rats in the model group were pale with clear cystic dilated cells on the surface,while those in the blank group were ruddy.HE staining: a certain number of corpus luteum and follicles at different development stages can be seen under the light microscope of normal rat ovaries.The structure of follicles is clear,and the structures of egg cells,egg mound,hyaline zone and radial crown can be seen.The granulosa cells in the follicles are multi-layered,mostly 8-9 layers.PCOS model rats had more cystic dilated follicles and atretic follicles in their ovaries,the number of luteal and developmental follicles decreased significantly,the number of oocytes and radiative coronae in the follicles disappeared,the granulosa cells died,the number of granulosa cell layers decreased or disappeared,and the follicular membrane cells and stromal cellsproliferated.The above results indicated that the model group was successful in modeling.2.Post-administration intervention evaluation:(1)General conditions: in the blank group,the rats were generally in good condition in terms of spirit,activity response,food intake and drinking water,etc.,while in the model group,the symptoms were still obesity,fatigue,sleepiness,decreased activity,and insensitive response to external stimuli.Daily food intake,water intake and urine volume were all greater than those in the blank group.The traditional Chinese medicine group and the western medicine group had good spirit and sensitive reaction,the food intake of western medicine group were less than those of traditional Chinese medicine group.(2)Weight of rats: after treatment,the weight of rats in the traditional Chinese medicine group and the western medicine group decreased significantly compared with that in the model control group(P<0.05),and the weight loss of rats in the western medicine group was more significant(P<0.05).(3)Estrus cycle: after treatment,the estrus cycle of the traditional Chinese medicine group and the western medicine group gradually recovered and became normal.The model control group still showed the disorder,prolonging or stagnation of the estrous cycle.(4)HOMA-IR,SHBG and FAI levels in rats: Bushen Tiaojing Decoction and metformin both effectively reduced homa-ir and FAI levels and effectively increased SHBG levels,which were statistically significant compared with the model control group(P<0.05).(5)Morphology of rat ovarian tissue: after treatment,follicles of different development stages and a certain number of corpus luteum were observed under the light microscope of the ovaries of rats in thetraditional Chinese medicine group and the western medicine group,and granulosa cells in the follicles were multilayered.In the model control group,the number of follicles,corpus luteum and granulosa cells still decreased significantly.(6)Expression of mir-21 in the ovaries of rats: the expression of mi R-21 in the ovaries of the traditional Chinese medicine group and the western medicine group was significantly higher than that of the model control group(P<0.05).Conclusion:1.The PCOS rat model can be successfully established by intragastric infusion of letrozole solution and feeding with high-fat diet by letrozole modeling;2.Bushen Tiaojing Decoction and Metformin both reduced serum HOMA-IR and FAI levels in PCOS model rats,while Bushen Tiaojing Decoction and Metformin both increased serum SHBG levels in PCOS model rats.3.Bushen Tiaojing Decoction and Metformin could improve the expression of mi R-21 in the ovarian tissues of PCOS model rats.4.The pathogenesis of PCOS may be related to hyperandrogen and insulin resistance.Abnormal expression or dysfunction of ovarian mi R-21 may affect follicular development and atresia,as well as hormone synthesis and metabolism.Bushen Tiaojing Decoction may effectively improve the follicular development and ovulation status of PCOS model rats by improving the phenomenon of hyperandrogen and insulin resistance,so as to effectively treat PCOS. |
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