To Explore The Molecular Biological Basis Of Endometrial Receptivity In Phlegm-dampness Block Polycystic Ovary Syndrome Based On MAPK/ERK Signaling Pathway

Objective:This subject adopts dehydroepiandrosterone（DHEA）preparation of polycystic ovary syndrome（PCOS）model in rats,after the success of the model respectively to pale with phlegm decoction,metformin to intervene,to observe the different intervention group and the receptivity of endometrium of rats markers LIF,expression of integrinαvβ3 levels of change and influence on the MAPK/ERK signaling pathway,In addition,the molecular biological basis of endometrial receptivity of phlegm-dampness block polycystic ovary syndrome was preliminarily discussed based on the MAPK/ERK signaling pathway by integrating the related sex hormone indexes and the histopathological changes of uterus and ovary.Methods:Forty-six SPF female SD rats at 21 days of age were purchased and divided into blank control group（n=13）and model group（n=33）by random number table method.The modeling lasted for 20 days.Rats in the modeling group were subcutaneously injected with DHEA（6mg/100 g/d+0.2m L soybean oil for injection）on the neck and back every day to prepare PCOS model rats,and rats in the blank control group were subcutaneously injected with 0.2ml soybean oil for injection every day.The estrous cycle,serum sex hormone levels and morphological changes of ovary were observed to determine the model formation of rats.After modeling,the rats were divided into model group,Cangfu Daotan Decoction group and metformin group by random number table method.The blank group and model group were given drinking water for gavage,and the latter two groups were given Cangfu Daotan Decoction and metformin for gavage intervention for 4 weeks.After intervention,the serum levels of follicle-stimulating hormone（FSH）,luteinizing hormone（LH）,testosterone（T）,estradiol（E₂）and insulin（INS）were detected by enzyme-linked immunosorbometry（ELISA）.HOMA insulin resistance index（HOMA-IR）was calculated.The morphological changes of ovary and endometrium were observed by hematoxylin-eosin（HE）staining.The m RNA expression levels of ERK1,ERK2,LIF and integrinαVβ3 in rat endometrial tissues were detected by real-time PCR.Western Blot and immunohistochemistry were used to detect the protein expression levels of ERK1,ERK2,P-ERK1,P-ERK2,LIF and integrinαVβ3 in rat endometrial tissues.Results:1.Comparison of various indicators after modeling:（1）Compared with blank control group,serum T and LH levels,INS and HOMA-IR levels of rats in modeling group were significantly increased（P<0.01）,FSH and E₂levels were significantly decreased（P<0.01）,there was no significant difference in FPG between the two groups（P>0.05）.（2）The ovaries of rats in the blank control group were bright red,while those in the model group were pale.（3）In the blank control group,ovarian follicles and multiple luteal bodies at different stages of development were observed under light microscope,and granulosa cells had complete morphology.In the model group,ovarian follicles showed cystic dilatation,follicles and luteum were rare,and the number of granulosa cell layers decreased.（4）After the modeling,the estrous cycle of rats in the blank control group was 4-5days and showed regular changes,while the modeling group lost the regular estrous cycle and remained in the interestrous or estrous phase.2.Comparison of indicators after intervention:（1）Comparison of serum sex hormone levels in each group:Compared with blank group,serum T and LH levels in model group were significantly increased（P<0.01）,E₂and FSH levels decreased significantly（P<0.01）.Compared with model group,serum T and LH levels in Cangfu Daotan Decoction group were significantly decreased（P<0.01）,E₂level and FSH level increased significantly（P<0.01）;Serum T,FSH and E₂levels in metformin group were not significantly different from those in model group（P>0.05）,LH level was significantly decreased compared with model group（P<0.05）.（2）Comparison of FPG and HOMA-IR index in each group:there was no sig nificant difference in FPG level among all groups（P>0.05）.Compared with blank g roup,serum INS and HOMA-IR levels in model group were significantly increased（P<0.01）.Compared with model group,the levels of INS and HOMA-IR in Cangf u Daotan Decoction group and Metformin group were significantly decreased（P<0.01 or P<0.05）.（3）Comparison of the morphological characteristics of ovarian tissues of rats in each group:the ovary of rats in the blank group was bright red under stereoscopic microscope,and multiple luteal follicles and granulosa cells at different stages of development could be seen under light microscope.The morphology of granulosa cells was complete and neatly arranged.Ovaries of rats in model group were pale in color under stereoscopic microscope,follicles showed cystic dilatation under light microscope,follicles and luteum were rare,granulosa cell layers were reduced and sparsely arranged.Compared with model group,cangfu Daotan Decoction group and metformin group were improved.（4）Endometrial histomorphological changes of rats in each group:Compared with blank group,endometrial thickness of model group was significantly decreased（P<0.01）.Compared with model group,the endometrial thickness of cangfu Daotan Decoction group was significantly increased（P<0.01）,there was no significant diff erence between metformin group and model group（P>0.05）.In the blank group,the re were more endometrial glands with large lumens and folds,and a large number of glands were distributed in clusters.Endometrial epithelial cells were arranged ne atly and rich in cytoplasm.In the model group,the number of glands decreased,an d most of them were single glands,sparsely arranged,and the lumen became small.The endometrial epithelial cells contracted in a low column shape and there were vacuoles in the cytoplasm.Both cangfu Daotan Decoction group and metformin gro up showed an increase in the number of glands,enlarged glandular lumen,orderly arrangement of endometrial epithelial cells and rich cytoplasm.Compared with metf ormin group,the glandular lumen of metformin group was enlarged but slightly str aight,and the amount of gland increase was not as obvious as that of Cangfu Dao tan decoction group.（5）The m RNA relative expression levels of ERK1,ERK2,LIF and integrinαvβ3 in each group:compared with blank group,m RNA expression levels of LIF and integrinαvβ3 in model group were significantly decreased（P<0.01）.Compared with model group,the mrna expression levels of LIF and integrinαvβ3 in Cangfu Daotan Decoction group were significantly increased（P<0.01）,metformin group also increased,but there was no significant difference between metformin group and model group（P>0.05）;There was no significant difference in the mrna expression levels of ERK1 and ERK2 among all groups（P>0.05）.（6）Comparison of LIF and integrinαvβ3 protein expression levels in all groups:compared with blank group,LIF and integrinαVβ3 protein expression levels in model group were significantly decreased（P<0.01or P<0.05）.Compared with model group,the expression levels of LIF and integrinαVβ3 protein in Cangfu Daotan Decoction group were significantly increased（P<0.01）,while there was no significant difference between metformin group and model group（P>0.05）.（7）Comparison of protein expression levels of ERK1,ERK2,P-ERK1and P-ERK2 in each group:Compared with blank group,the relative protein expression levels of P-ERK1and P-ERK2 and the ratio of P-ERK1/ERK1 and P-ERK2/ERK2 in endometrial tissues of model group were significantly increased（P<0.01）.Compared with model group,the relative protein expression levels of P-ERK1 and P-ERK2 and the ratio of P-ERK1/ERK1and P-ERK2/ERK2 in endometrial tissues of cangfu Daotan Decoction group were significantly decreased（P<0.01）.There were significant differences in P-ERK2 expression and P-ERK2/ERK2 ratio between metformin group and model group（P<0.01）,but there were no significant differences in P-ERK1 expression and P-ERK1/ERK1 ratio between model group and model group（P>0.05）.There was no significant difference in the relative expression levels of ERK1 and ERK2 proteins among all groups（P>0.05）.Conclusions:1.Dampness and phlegm removal can improve the levels of related sex hormones in PCOS rats and also have a positive effect on the morphological changes of ovary and uterus.2.Dampness and phlegm-reducing can improve endometrial receptivity of PCOS by regulating m RNA and protein expression levels of ERK1,ERK2,LIF and integrinαVβ3 in endometrial tissue.3.Decreased endometrial receptivity caused by abnormal MAPK/ERK signaling pathway may be one of the biological bases of PCOS phlegm-dampness block syndrome.