Study On The Mechanism Of Improving Endometrial Receptivity In Polycystic Ovary Syndrome By Acupuncture Based On “Chong Channel Being Sea Of Blood” Via VEGF/PI3K/AKT Mediated Angiogenesis

Objective.On the basis of in-depth analysis of the theoretical basis of " Chong channel being sea of blood ".To explore the effect mechanism of " Chong channel being sea of blood " acupuncture therapy to improve angiogenesis and endometrial receptivity in polycystic ovary syndrome from the VEGF/PI3K/AKT pathway,and to provide new ideas and strategies for the application of acupuncture in the reproductive field.Methods.1.We collated and analyzed ancient medical literature on " Chong channel being sea of blood ",thus provided a theoretical basis for the acupuncture based on " Chong channel being sea of blood.We also analyzed the " Chong channel being sea of blood "acupuncture created by Professor Zhao Rong and analyzed its clinical efficacy in the field of female reproduction.2.Bioinformatically identified and analyzed the differences in endometrial gene expression profiles between patients with polycystic ovary syndrome and infertility,performed GO and KEGG analysis on the differential genes,created PPI networks and performed signaling pathway screening,and finally identifed Hub genes.3.We used dehydroepiandrosterone,letrozole and estradiol to construct three models of polycystic ovary syndrome,and analyzed and compared the changes of motility cycle,serum sex hormones,ovarian pathological morphology and body weight growth rate of rats in different models to select the best model of polycystic ovary syndrome.We also improved the animal study protocol of "Chong channel being sea of blood".4.The disease model of dehydroepiandrosterone-induced polycystic ovary syndrome was constructed and randomly divided into control group,model group,acupuncture group and PI3 K inhibitor + acupuncture group.After acupuncture or inhibitor intervention,the general state,body weight growth rate,organ index,estrous cycle,gross observation of pregnancy outcome(number of rat supposition and embryo implantation),ELISA assay of serum sex hormone index(E2,T,LH,FSH),HE staining assay of ovary and uterus morphology(endometrial thickness and number of blood vessels),scanning electron microscope observation were observed and compared.The endometrial surface pinocytosis of rats during the "implantation window" was observed,and the endometrial receptivity indexes(HOXA10 and LIF protein expression)were detected by Western Blot.5.Based on bioinformatics analysis of key signaling pathways,we further used q PCR and Western Blot to detect the transcription and protein expression of molecules related to VEGF/PI3K/AKT signaling pathway,and immunohistochemistry to detect the expression and localization of VEGF and Ang-1 for vascular neovascularization,and validate the mechanism based on " Chong channel being sea of blood ".The mechanism of VEGF/PI3K/AKT pathway regulation by acupuncture to promote endometrial angiogenesis in PCOS and thus improve endometrial receptivity and pregnancy outcome.6.The EPCS-related mobilization cytokines(VEGF,PDGF)in serum were detected by ELISA,endometrial EPCS homing(CD34+CD133+VEGF+triple staining)was detected by immunofluorescence,and VEGF/PI3K/AKT signaling pathway downstream endothelial progenitor cell mobilization and homing pathway-related molecules were detected by q PCR and Western Blot.To investigate the mechanism of promoting endothelial progenitor cell homing through HIF-α/SDF-1α/CXCR4 by acupuncture based on " Chong channel being sea of blood " to improve endometrial angiogenesis in PCOS rats.Results.1.Literature analysisThe theoretical basis of " Chong channel being sea of blood " is closely related to the naming,origin and physiological functions of the Chong Vessel.The acupuncture therapy based on " Chong channel being sea of blood " founded by Prof.Zhao Rong uses Gong Sun and Neiguan as the main acupuncture points,and adopts corresponding matching acupuncture points and acupuncture methods according to the staging of women’s menstrual cycle to regulate the Qi and Blood of the Chong Vessel,nourish the uterus,alleviate sex hormone disorders in infertile women,improve endometrial receptivity and angiogenesis,and ultimately improve pregnancy outcomes.The clinical efficacy is remarkable.2.Key genes and pathways of polycystic ovary syndrome and infertilityA total of 41 differentially expressed genes were screened for polycystic ovary syndrome and infertility,of which 29 differentially expressed genes were up-regulated and12 differentially tagged down-regulated.The main biological process of GO enrichment was muscle contraction,the main cellular component was tight junction,and the main molecular function was vitamin formate synthesis;KEGG pathway enrichment was mainly in TNF signaling pathway,cell adhesion molecules,tight junction,VEGF signaling pathway,etc.The PPI results showed that multiple complex pathways can interact with each other in the pathogenesis of polycystic infertility,and 10 hub genes were screened: CDC6,CCNB1,CDK2,CCND1,CDKN1 B,CDKN3,CCND2,CDC25 B,CCNA1,CDC25 B,CDC25B,CCNA2,CDC25 B,CDC25B,CDC25 B,CDC25B,CDC25 B,CDC25B,CDC25 B,CDC25B,CDC25 B,CDC25B,CDC25 B and CDC25 B.All of the above screened pathways and genes can be involved in the construction of endometrial receptivity to varying degrees and are closely related to angiogenesis.3.Study on the mechanism of acupuncture to improve endometrial receptivity and angiogenesis in PCOS based on " Chong Channel being Sea of Blood".(1)Establishment and evaluation of SD female rats with polycystic ovary syndrome model: Compared with the control group of rats,all three groups of model replication method induced typical pathological features such as motility cycle disorder,weight gain,sex hormone disorder and abnormal follicle development.Among them,the pathology of the dehydroepiandrosterone group showed polycystic-like changes with significantly increased serum sex hormone T and E2 levels(P<0.01),increased LH and LH/FSH ratios(P<0.05)and decreased FSH levels(P<0.05),which were most consistent with PCOS,so the DHEA regimen was chosen to induce polycystic ovary syndrome in the subsequent efficacy evaluation and mechanism study.(2)Modified the protocol of acupuncture therapy based on "Chong channel being sea of blood" in animal experiments: using the tie-band cage fixation method,using Gongsun and Neiguan as the main acupoints,together with Zi Gong,Guan Yuan and Qi Chong,and adding abdominal electroacupuncture with 2 Hz continuous wave,with mild local muscle tremor,current intensity of 1 m A,and duration of 20 min,which was determined as the protocol for animal experiments.(3)Study on the improvement of polycystic-like symptoms,embryonic implantation and endometrial receptivity in PCOS rats by "Chong channel being sea of blood” based acupuncture: compared with the control group,the rats in the model group had increased body weight and ovarian index(P<0.05),disturbed motility cycle,significantly increased levels of sex hormones E2 and LH(P<0.01),increased levels of LH/FSH and T(P<0.05)and decreased levels of FSH(P<0.01),abnormal ovarian pathology,significantly increased atretic follicles and cystic dilated follicles,and significantly decreased corpus luteum.In terms of endometrial receptivity,endometrial thickness and neovascularization decreased(P<0.05),pinopodes showed atrophy and degeneration,and the expression of HOXA10 and LIF,molecular markers of endometrial receptivity,decreased significantly(P<0.01),and the number of final embryo implantation sites decreased significantly(P<0.01).After acupuncture intervention,compared with the model group,the weight growth rate and ovarian coefficient were significantly reduced(P<0.05),the motility cycle was restored,E2,T and LH levels were significantly reduced(P<0.01),LH/FSH ratio was reduced(P<0.05),FSH levels were significantly increased(P<0.01),ovarian pathological morphology was restored,and ovarian tissue granulosa cell layer and corpus luteum were increased.In terms of endometrial receptivity: increased endometrial thickness(P<0.05),significantly increased the number of neovascularization(P<0.01),good morphology and higher number of pinopodes,significantly increased HOXA10 and LIF expression both(P<0.01),and finally significantly increased the number of embryonic implantation sites(P<0.01).However,the combination of PI3 K inhibitor LY294002 with acupuncture reversed the above efficacy of acupuncture,suggesting that acupuncture based on " Chong channel being sea of blood" can improve the symptoms of polycystic ovaries in PCOS,endometrial receptivity and embryo implantation site number during the window of implantation in rats,and the mechanism is related to PI3K/AKT pathway.(4)Study on the improvement of endometrial angiogenesis through VEGF/PI3K/AKT in PCOS rats by " Chong channel being sea of blood " based acupuncture: compared with the control group,the immunohistochemical results showed that the expression of VEGF and Ang-1,the indicators of endometrial angiogenesis in the PCOS model group during the window of implantation,decreased(P<0.05),WB results showed that the expression of VEGF,P-PI3 K,AKT and P-AKT in the pathway was decreased(P<0.05),and the expression of VEGFR2 and AKT was significantly decreased(P<0.01).q PCR results showed that the relative expression of Vegf,Vegfr,Pi3 k,Akt m RNA was significantly decreased(P< 0.01),ELISA results showed a significant decrease in e NOS and NO expression downstream of the pathway(P<0.01),suggesting a decrease in endometrial angiogenesis and VEGF/PI3K/AKT pathway expression in PCOS rats.After acupuncture intervention,it significantly increased the expression of VEGF and Ang-1,indicators of angiogenesis(P<0.05),increased VEGF,P-PI3 K,P-AKT protein expression(P<0.05),significantly increased VEGFR2,AKT protein expression(P<0.01),significantly increased Vegf,Pi3 k,Akt m RNA expression(P<0.01),elevated Vegfr m RNA expression(P<0.05),and increased e NOS and NO expression downstream of the pathway(P<0.01).The PI3 K inhibitor reversed the above mentioned efficacy of acupuncture,which is evidence that acupuncture based on " Chong channel being sea of blood" promotes angiogenesis by activating VEGF/PI3K/AKT.(5)Study on the improvement of endometrial angiogenesis in PCOS rats by promoting endothelial progenitor cell homing through VEGF/PI3K/AKT with " Chong channel being sea of blood " based acupuncture: compared with the control group,the serum endothelial progenitor chemokines VEGF and PDGF were significantly decreased in the PCOS model group(P<0.01);for EPCs homing,the immunofluorescence results showed that the positive rate of endothelial progenitor-specific marker CD34+CD133+ double-labeled cells was significantly decreased in the model group(P<0.01),and the positive rate of VEGFrecruiting EPCs homing i.e.CD34+CD133+VEGF+ triple-labeled cells was significantly decreased(P<0.01);for pathways,WB results showed that the relative protein expression of HIF-1α and SDF-1 was decreased in the model group(P<0.05),and the relative protein expression of CXCR4 was significantly decreased(P<0.01).q PCR results showed that Hif-1α,Sdf-1,and Cxcr4 m RNA expression were significantly decreased in the model group(P<0.01),suggesting that endothelial progenitor cells in PCOS rats mobilize.The endothelial progenitor cells of PCOS rats had decreased chemokine mobilization and impaired nesting to the endometrium.After the acupuncture intervention based on "Chong channel being sea of blood",compared with the model group,acupuncture significantly increased the levels of serum chemokines VEGF and PDGF(P<0.01),significantly increased the positive rate of CD34+CD133+ double-labeled cells(P<0.01),and significantly increased the expression of CD34+CD133+ triple-labeled cells(P<0.01).CD133+VEGF+ triple-labeled cells positive rate(P<0.01);increased HIF-1α and SDF-1 expression(P<0.05),significantly increased CXCR4 protein expression(P<0.01),increased Hif-1α and Sdf-1 m RNA expression(P<0.05),significantly increased Cxcr4 m RNA expression(P<0.01),PI3 K inhibitors reversed the above mentioned efficacy of acupuncture,which in turn proved that acupuncture based on " Chong channel being sea of blood" could activate VEGF/PI3K/AKT pathway,increase downstream HIF-α/SDF-1α/CXCR4 key pathway protein and m RNA expression,increase chemokine.It also increased the expression of chemokines,improved the mobilization of EPCs to the endometrium,promoted angiogenesis,and subsequently improved endometrial receptivity and pregnancy outcome in PCOS rats.Conclusion.1.The theory of " Chong channel being sea of blood" has a sound theoretical basis;the acupuncture based on " Chong channel being sea of blood" founded by our supervisor Prof.Zhao Rong,using Gongsun and Neiguan as the main acupuncture points,can effectively improve the sex hormone disorder of infertile women,increase the endometrial receptivity and angiogenesis,and ultimately improve the pregnancy outcome.2.Bioinformatics analysis screened key pathways of polycystic ovary syndrome and infertility,Hub genes can all be involved in the construction of endometrial receptivity to varying degrees,which is closely related to angiogenesis,suggesting that endometrial receptivity can be the entry point for infertility in PCOS.3.VEGF/PI3K/AKT was selected for experimental validation,and the results showed that the acupuncture based on " Chong channel being sea of blood " significantly alleviated the polycystic-like symptoms,improved endometrial receptivity and increased embryonic implantation sites in PCOS rats,and the underlying mechanism may be as follows:acupuncture based on "Chong as the Sea of Blood" could activate the VEGF/PI3K/AKT pathway,and increase the downstream HIF-1α/SDF-1α/CXCR4 key pathway protein and m RNA expression,improve the mobilization of EPCs to the endometrium,promote angiogenesis,and subsequently improve the endometrial receptivity and pregnancy outcome in PCOS rats.