| Objective In this study,the female athlete triad(FAT)in female SD rats were established,and Jianpi Bushen decoction was used to prevent and treat rats with FAT.Discuss the effects of Jianpi Bushen decoction on the morphology and function of female reproductive system and bone tissue by observing the basal indexes,organ pathological changes and hormone level changes.We investigated the correlation between the pathological mechanism of hypothalamic-pituitary-ovarian(HPO)axis and the mechanism of its protection by Jianpi Bushen prescription,and the transforminggrowth factor-β(TGF-β)/Smad(R-Smad/Smad)signaling pathway at the molecular level,which provided an experimental basis for the prevention and treatment of FAT by Chinese medicine.Methods1: The establishment of FAT model ratsNinety healthy female rats,12 weeks old,SPF class,80-220 g were fed for one week and swam adaptively for 3 days.The 3 rats that could not swim or had great change were excluded,and then the rest were randomly divided into control group(n=15),model group(n=15),Jianpi group(n=15),Bushen group(n=15),Jianpi Bushen group(n=15),and positive drug group(n=15)according to the random number table.The rats in control group were reared normally.The other groups of rats were all fasted alternately for 13 days and then swam with 3%-5% weight loading.The rats swam without weight for 10 min on first day,then increased 1% weight for each week,and swam with exhaustion,3% weight for the first week,4% weight for the second week,and increased to 5% weight for the third week,and continued this exercise intensity until the end of 7 weeks.Behavioral indexes such as swimming time,skin,hair,and behavioral activities of rats in each group were observed,as well as changes in body weight and morphological changes of vaginal mucous membrane cells of rats in each group.Anatomical morphology and pathological changes of uterus and ovary of rats in each group were observed,uterine index was calculated,and the contents of estradiol,progesterone,calcium and phosphorus in serum of rats were detected.2: Study on the mechanism of Jianpi Bushen decoction for FAT model ratsAfter 13 days of alternate fasting,all the 6 groups of rats were observed according to their each protocols.In the model group,increasing weight-bearing swimming exercise and restricted diet were used for gavage with distilled water for 7 weeks;In the Jianpi group,increasing weight-bearing swimming exercise and restricted diet was given concentrated medicine juice of Jianpi decoction(Sijunzi decoction)by gavage for a total of7 weeks;In the Bushen group,increasing weight-bearing swimming exercise and restricted diet was given concentrated medicine juice of Bushen decoction(Liuwei Dihuang decoction)by gavage for a total of 7 weeks;In the Jianpi Bushen group,increasing weight-bearing swimming exercise and restricted diet was given concentrated medicine juice of Jianpi Bushen decoction(Sijunzi +Dihuang decoction)by gavage for a total of 7weeks;In the positive drug group,increasing weight-bearing swimming exercise and restricted diet,and received estrogen injection for a total of 7 weeks.Start give dosing a week before exhausting swimming.HE staining was used to observe the morphological changes of the exhaled cells of the vaginal mucosa.On the last day,bone density of the whole body of the rats were measured by X-ray bone densitometer.On the second day after exhaustive swimming,the anatomical morphology and pathological changes of the uterus and ovary of the rats in each group were observed,and the uterine indexes were calculated.The contents of estradiol 2,progesterone calcium and phosphorus in rat serum were detected.The protein expression levels of TGF-β1,Smad4,p-Smad2 and p-Smad3 in TGF-β/Smad signaling pathway were detected by immunohistochemical method.Results1.Establishment of FAT model rats1.1 Body weight: Compared with the blank group,the body weight of rats in model group decreased significantly(P<0.05).1.2 Observation of vaginal exfoliated cells: Compared with the control group,the smears of vaginal exfoliated cells of rats in the model group showed the reduction of nuclear epithelial cells and keratinocyte,and the estrous cycle was significantly prolonged and irregular,and even showed the interestrous period for a long time.The estrous cycle of rats in blank group was generally in the range of 4-5 days,while that of rats in model group was in the range of 8-9 days,P<0.05.In model groups,the estrous cycles were irregular for 3and 11 days.1.3 Serum estradiol,progesterone contents : Compared with control group,the contents of estradiol,progesterone in serum of rats in model group were significantly decreased(P<0.05).1.4 Bone mineral density detection: Compared with control group,the bone mineral density of rats in model group was significantly decreased.1.5 Serum calcium,phosphorus contents: Compared with control group,the contents of calcium in serum of rats in model group were significantly decreased(P<0.05);Phosphorus content was significantly increased(P<0.05).2.Study on the mechanism of Jianpi Bushen prescription for FAT model rats2.1 Exhaustive swimming time: Compared with model group,exhaustive swimming time of rats in Jianpi group and Jianpi Bushen group was prolonged,but the difference was not significant(P>0.05);Exhaustive swimming time of rats in,Bushen group was significantly prolonged,P<0.05.2.2 Body weight and drinking water :Compared with model group,the body weight of rats in Jianpi group group was slightly increased(P<0.05),and the body weight of rats in,Bushen group was the same as that in Jianpi Bushen group group(P<0.01),showing significant difference.Compared with model group,the drinking water volume of the TCM group was significantly increased,while the positive drug group was equivalent,with no significant difference,P> 0.05.2.3 Histopathological observation of uterus and ovary: Compared with model group,irregular lesions of uterus and ovary in administration group were less and a significant increase in ovarian growth follicles.2.4 Observation of vaginal shedding cells: Compared with the rats in the model group,the number of vaginal shedding cells in the four early,early,late and interphase increased significantly;and the estrous cycle was significantly prolonged,there was a significant difference,P <0.05.2.5 Contents of estradiol,progesterone,calcium and phosphorus in serum: Compared with model group,the contents of E2,progesterone P and calcium Ca in Jianpi group,Bushen group,and Jianpi Bushen group were significantly increased,P<0.05;P content was significantly decreased,P<0.05.2.6 Pathological observation of femur and tibia: Compared with control group,bone trabeculae in bone tissue of rats in model group were unevenly distributed,with more osteoclasts and congestion points.Compared with the model group,bone tissue composition increased in the administration group,bone trabeculae were sparse in the Jianpi group and Bushen group,and the degree of bone thinning and fracture was reduced.The structure of Jianpi Bushen group was more complete and continuous.2.7 Expression of proteins related to TGF-β /Smad signaling pathway: Compared with control group,contents of TGF-β 1 and Smad4 in model group were significantly decreased,P<0.05.Compared with model group,the content of TGF-β1 in Jianpi group was slightly increased(P<0.05),while the content of Smad4 was slightly increased(P>0.05).The content of TGF-β1 in Bushen group and invigorating spleen and tonifying kidney group was not significantly increased,P>0.05;The content of Smad4 in Bushen group and Jianpi Bushen group was significantly increased P<0.05.Conclusion1.The modeling method of 13-day alternating fasting and exhaustive swimming with3%-5% weight can cause three major symptoms of reduced available energy,irregular menstruation and osteoporosis in rats,indicating that the animal models of FAT have been successfully constructed,and the modeling effect is good,with higher survival rate and success rate.2.The occurrence of the triad of female athletes is related to the hypothalamic-pituitary ovarian axis,resulting in irregular changes in the morphology of uterus and ovary,decreased uterine index and prolonged estrous cycle.Femur,tibia osteoporosis,bone density decrease.The contents of estradiol E2,progesterone P and calcium Ca in the body were reduced,and the contents of P increase.3.Jianpi Bushen prescription can improve three symptoms of the triad of female athletes and make them gain weight;Uterine and ovarian lesions decrease,uterine index increase,estrous cycle decrease;osteoporosis and bone density reduction in femur and tibia are improve;The contents of estradiol E2,progesterone P and calcium Ca in the body increase,while the content of P decrease.4.FAT may be caused by TGF-β/Smad signaling pathway,Jianpi Bushen prescription can inhibit Smad2 and Smad3 phosphorylation,promote the generation of Smad4,reduce the expression of TGF-β1,reduce the occurrence of FAT. |
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