| Background and purpose of the research:The survey showed that the incidence of infertility was 10-12%in our country,and the incidence of infertility with stunted follicle was increasing year by year.Follicular dysplasia refers to the dysfunction of the HPO axis and the paracrine/secretory function of the ovary,which leads to follicular non-development,developmental arrest,atrexia,ovulation of immature follicles,etc.,thus leading to infertility.In recent years,the incidence of this disease is on the rise and has become one of the important factors affecting female fertility.Kisspeptin is a peptide hormone encoded by KISS-1 gene.By binding with its receptor GPR54,Kisspeptin induces the pulse release of GnRH,regulates the HPO axis,and is involved in regulating the growth and development of the reproductive system and the secretion of reproductive hormones.Kisspeptin has gradually become a research hotspot of infertility related diseases with follicular developmental disorders such as PCOS,DOR and POF.Kp/GPR54 system is not only distributed in the central nervous system,but also expressed in the peripheral ovaries,which can regulate ovarian function and follicle development,promote oocyte maturation,and induce ovulation.The treatment of follicular dysplasia in western medicine is mainly to promote ovulation with drugs,but there are many adverse reactions.Professor Xiao Chengcheng thought that deficiency of kidney and Chong Ren disorder were the fundamental mechanism of follicle developmental disorder.In treatment,Qizi Yishen Li Chong Decoction was used as the basic principle.Our previous study found that Qizi Yishenlichong Decoction can improve follicular development in PCOS rats,and its mechanism may be related to the effect of Kp/GPR54 system on GnRH neuron activity and the regulation of HPO axis.Based on this,the purpose of this study was to construct a rat model of follicular dysplasia,and to observe hormone levels and ovarian histommorphology of rats in each group by Qizi Yishenlichong Decoction as intervention means,so as to explore the role of Kp/GPR54 system and its signaling pathway in follicular dysplasia,hoping to provide new ideas for the early prevention and standard diagnosis and treatment of this disease.Research methods:1.This study describes the research progress of Kp/GPR54 system and follicular dysplasia,as well as the understanding of etiology,pathogenesis and treatment of follicular dysplasia in traditional Chinese medicine.In summary,Professor Xiao reviewed his academic thought on the treatment of infertility caused by developmental follicle disorder,and analyzed the clinical experience of Professor Xiao by combining a classic clinical case.2.48 female SD rats aged 8-10 weeks with regular estrous cycles were randomly divided into 6 groups by random number table method,including blank group,model group,western medicine group,high dose group,medium dose group and low dose group,with 8 rats in each group.Except for blank group,the rats in other groups were given 50mg/(kg·d)of Tripterygium wilfordii polyglycoside tablet suspension for 3 consecutive estrous cycles to establish a rat model of follicular dysplasia.After successful modeling,the blank group and model group were given 10 ml/(kg·d)sterile drinking water intragastric administration,the western medicine group was given sequential treatment with estradiol valerate and medroxyprogesterone acetate,and the Chinese medicine groups were given high,medium and low doses of Qizi Yishenlichong Tang granules intragastric administration,once a day,for 3 consecutive estrous cycles(15d).After the intervention,the general behavioral changes of the rats in each group were observed,the ovarian wet weight and ovarian coefficient of the rats were compared,the appearance of the rats in each group was observed by stereomicroscope,the histological changes of the rats in the ovary were observed by HE staining,and the serum Kp,AMH and INHB levels were detected by enzyme-related immunosorbent assay.The expression levels of Kp,GPR54,extracellular signal-regulated kinase 1/2(ERK1/2),p38 and JNK in ovary of rats in each group were detected by Western-blot.The mRNA expression levels of Kp,GPR54,ERK1,ERK2,p38 and JNK in rat ovarian tissue were detected by real-time quantitative PCR(RT-PCR).Research results:1.Comparison of the general behavior of the rats in each group:the rats in the blank group were in a good mental state,with high activity,white,smooth and moist fur,no hair shedding and back bending,free movement,normal drinking and eating,normal urination,and good stool shape;Compared with the blank group,the rats in the model group showed obvious listless spirit,decreased activity,crouching,coarse and sparse fur,dull and dull hair color,local hair loss,continuous aggravation of the back,reduced food intake,increased water intake,loose stool,distinct odor,and frequent urination after gavage with tripterygium wilfordii.Compared with the model group,the western medicine group and the Qizi high-dose and medium-dose groups showed improved behavior,improved mental state,increased activity,hair color gradually recovered moist,hair removal and back arch no longer increased,diet and second stool gradually returned to normal,while the Qizi low-dose group showed a general mental state,coarse hair,slow improvement of hair removal and back arch,no obvious increase in food intake,and thin stool texture.2.Comparison of ovarian wet weight and ovarian coefficient among all groups:There were no significant differences in ovarian wet weight among all groups(P>0.05),Compared with blank group,the ovarian coefficient in model group,Qizi medium and low dose groups was decreased(P<0.05).After drug intervention,compared with model group,the ovarian coefficient in western medicine group and Qizi high-dose group was increased(P<0.05).3.Comparison of the appearance and histology of the ovary of rats in each group:Under stereomicroscope,the appearance of the ovary of rats in the blank group was red in color,with clear outline of the follicles,orderly arrangement,and corpus luteum on the surface.Ovary volume atrophy,surface shrinkage,pale color in model group;Compared with the model group,the surface color of the ovary in Qizi high-dose and medium-dose groups was fresher and the follicle growth was regular,similar to that in the western medicine group,while the ovary structure was dense and the ovarian parenchyma was reduced in the low-dose group.The comparison of ovarian histopathology among the groups showed that the structure of the ovarian cortex and medulla in the blank group was clear,and multiple follicles at different developmental stages were observed,with active growth,full oocytes,numerous granular cell layers,dense and orderly arrangement,corpus luteum distribution,and few atretic follicles.In the model group,the number of ovarian follicles at all levels was reduced,and the atretic follicles were more common,the cavity was enlarged,the number of granular cell layers was reduced,and the arrangement structure was disordered.In the western medicine group and Qizi Yishen Lichong decoction high-dose and medium-dose groups,the ovarian tissue morphology was improved,the structure of follicles at all levels was clear,the dominant follicles were visible,the number of granulosa cell layers was increased,the atretic follicles were significantly reduced,the cavity was narrowed,and the corpus luteum was visible.In the low-dose group,some ovarian follicles were poorly developed,secondary follicles were rare,the granulosa cell layer was thin,and the cavity shrinkage of atresia follicle was not obvious.4.Comparison of serum Kp,AMH and INHB levels in each group:Compared with blank group,serum Kp,AMH and INHB levels in model group and Qizi low-dose group were significantly decreased(P<0.05),serum AMH and INHB levels in Qizi medium-dose group were significantly decreased(P<0.05),and serum INHB levels in western medicine group were significantly decreased(P<0.05).After drug treatment,compared with model group,serum Kp,AMH and INHB levels in western medicine group and Qizi high-dose group were significantly increased(P<0.05),and serum AMH and INHB levels in Qizi medium-dose group were significantly increased(P<0.05);The serum levels of Kp,AMH and INHB in western medicine group and Qizi high-dose group were higher than those in Qizi low-dose group(P<0.05).Correlation analysis showed that Kisspeptin,AMH and INHB were positively correlated(P<0.01).5.Comparison of relative protein expression levels of Kp,GPR54,p-ERK1/2/ERK1/2,p-p38/p38 and p-JNK/JNK in ovarian tissue of rats in each group:Compared with blank group,the relative protein expressions of Kp,GPR54,p-ERK1/2/ERK1/2,p-P38/p38 and P-JNK/JNK in ovary of rats in model group were significantly decreased(P<0.01).After drug intervention,compared with model group,the relative protein expressions of Kp,GPR54,p-ERK1/2/ERK1/2,p-P38/p38 and P-JNK/JNK in Qizi high-dose group were increased(P<0.05).The protein expressions of Kp,GPR54,p-ERK1/2/ERK1/2 and P-P38/p38 in western medicine group and Qizi medium-dose group were increased(P<0.01),while there were no significant differences in the protein expressions of each index in Qizi low-dose group compared with model group(P>0.05).Compared with western medicine group and Qizi high-dose group,protein expressions of Kp,GPR54 and P-P38/p38 in Qizi medium-dose and low-dose groups were decreased(P<0.05).6.Comparison of mRNA expression levels of Kp,GPR54,ERK1/2,p38 and JNK in ovarian tissue of rats in each group:Compared with blank group,mRNA expression levels of Kp,GPR54,ERK1/2,p38 and JNK in ovary of model group and seven sons low-dose rats were significantly decreased(P<0.01);After drug intervention,compared with model group,mRNA expression levels of Kp,GPR54,ERK1/2,p38 and JNK in ovary of Qizi high-dose group were increased(P<0.01).The mRNA expression levels of GPR54,ERK1/2,p38 and JNK in ovary of western medicine group were increased(P<0.05,and GPR54,ERK1/2 and JNK in ovary of Qizi medium dose group were increased(P<0.05).There was no significant difference in mRNA expression between Qizi low-dose group and model group(P>0.05).Comparison between drug intervention groups showed that the expression levels of Kp,GPR54,ERK1/2,p38,and JNK mRNA in the ovaries of rats in the high-dose group of Qi Zi were higher than those in the low-dose group of Qi Zi(P<0.05 or P<0.01),while the expression levels of GPR54 mRNA in the ovaries of rats in the Western medicine group were higher than those in the medium and low-dose groups of Qi Zi and lower than those in the high-dose group of Qi Zi(P<0.05 or P<0.01).Conclusion:1.Qizi Yishenlichung Decoction with high and medium doses and sequential treatment with estrogen can improve the general behavior and ovarian coefficient of the model rats with follicular dysplasia,promote the recovery of ovarian appearance and ovarian histology,increase the number of follicles and luteum at all levels,increase the number of granulosa cell layers,reduce atretic follicles,and increase the expression levels of Kp,AMH and INHB in serum.It can reverse reproductive damage caused by tripterygium wilfordii polyglycoside tablets,promote follicle maturation and improve ovarian function.2.One of the targets of Qizi Yishenlichong Decoction in improving follicular development is the Kp/GPR54 system,which can effectively activate its downstream MAPK signaling pathway,especially inducing the phosphorylation of MAPK kinase such as ERK1/2 and p38,so as to play the role of regulating HPO axis,regulating reproductive endocrine,improving follicular growth and development,and protecting ovarian function. |
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