| PART Ⅰ Clinical ResearchEffect of Bushen Huoxue Recipe on uterine artery resistance and formation and degradation-related factors of ECM in menstrual blood of the patients with intrauterine adhesionsObjective:1.to research the feasibility of using endometrium derived from menstrual blood to study the mechanism of intrauterine adhesions;2.To evaluate the intervention effect of Bushen Huoxue Recipe on intrauterine adhesions(IUAs)patients in the process of uterine cavity repair by observing the changes in the expression levels of ECM formation and degradation-related factors,and PI3K,pAkt mRNA in menstrual blood of patients with IUAs.Methods:1.1:1 Individual matching paired design was used,40 IUAs patients in IUAs group and 40 healthy women in the control group were selected.Menstrual blood of all groups were collected on the second day of menstrual cycle.The expression of MMP-9、TIMP-1、COLIA1、TGF-β1,VEGF mRNA was detected in the sediment of the menstrual blood by RT-PCR.2.71 patients due to IUAs were selected and randomly divided into Chinese medicine group and control group.After Transcervical resection of adhesion,The control group of 36 patients were treated with oral Estradiol valerate and dydrogesterone,while the TCM group of 35 patients were treated with Bushen Huoxue Recipe and also combined with oral Estradiol valerate and dydrogesterone.The patients of these two groups,whose menstrual blood was collected on both the second or third day of the menstrual cycle before operation and the third menstrual cycle after operation be detected the expression levels of MMP-9,TIMP-1,COL1A1,VEGF,TGF-β1,PI3K,pAkt mRNA by qRT-PCR.The recovery of menstrual volume and the changes of TCM syndromes were observed before and after treatment,The endometrial thickness of these two groups of patients was measured under vaginal ultrasound on ovulation day,and the uterine artery resistance index RI was calculated.Results:1.1 There were epithelial and stromal cell components of endometrial cells in the menstrual tissue of IUAs normal patients.1.2 The mRNA level of MMP-9 and MMP-9/TIMP-1 in the IUAs group were significantly lower than those in the normal group.The mRNA level of TGF-β1 in the IUAs group were higher than those in the normal group,and there was statistical different between the IUAs group and the normal group.the expression of MMP-9/TIMP-1 was negatively correlated with the IUAs’ degree,and the expression of COLIA1、TGF-β1 was positively correlated with the degree of IUAs,P<0.05.2.1 After the TCRA and treatment,the menstrual volume of 20 cases(28.2%)returned to normal,the menstrual volume of 59 cases(83.1%)improved,the total effective rate was 93.0%.But after the intervention,there was no significant difference in menstrual volume between the two groups.Compared with the control group,the total score of TCM syndrome in TCM group declined with statistical difference(P<0.05).2.2 After the TCRA and treatment,the endometrial thickness of the Chinese medicine group was thicker than that of the control group with ultrasound evaluation,and the uterine artery resistance index RI of the TCM group was calculated,showed lower than that of the control group,with significant differences(P>0.05).2.3 The relative expression mRNA levels of MMP-9,VEGF,PI3k,pAkt in menstrual blood tissue of the TCM group were significantly much higher than those of the control group(P<0.05),but there was no significant difference in the expressions of TIMP-1,COL1A1 TGF-β1 mRNA between the two groups(P>0.05).Conclusion:1.There were epithelial and stromal cells in menstrual blood tissue,The decreasing expression of MMP-9、MMP-9/TIMP-1 and the increase of COLIA1、TGF-β1 in blood tissue of IUAs patients,which related with severy degree of IUAs,and maight paticipate in the formation of the IUAs.2.The Bushen Huoxue Recipe,which was Professor Qi Cong’s experience prescription,has the effect of Removing blood stasis and promoting regeneration by tonifying kidney had and essence.BSHX Recipe had the effect of increasing the endometrial thickness of postoperative IUAs patients,improvivg the endometrial blood flow,by promoting the expression of MMP9 and PI3K,pAkt in menstrual blood tissue and inhibiting extracellular matrix deposition.but also it could obviously promote the recovery of patients after operation and the prevention of IUAS again.PART Ⅱ Experimental ResearchThe Molecular Mechanism of Bushenhuoxue Recipe promoting the repair of the IUAs rats’Injured endometrium through OCT4-Mediated PI3k/Akt Pathway in endometrial injuryObjective:To evaluate the mechanism of BSHX Recipe promoting the repair of the IUAs rats’ Injured endometrium,by observing the Pathological endometrial changes of IUAs rats after intervention with BSHX Recipe and the related protein-expression of P13K/Akt/Oct4 signaling pathway in endometrial injury.Methods:The IUAs model rats were established by the model of double injury caused by mechanical and infection factors.The rats were divided into 4 groups:the normal group,the model group,BSHX group and Progynova group.The pathological changes of endometrium were observed by HE,The number of embryo implantation sites was calculated,and the Scanning electron microscope were used to observe the endometrial ultrastructure and pinodes.Immunohistochemical staining,RT-PCR and Western blot were adopted to detect the levels of MMP9,TGF-β1,VEGF related molecules and proteins,and the expression levels of PI3k/Akt pathway related factors and Oct4 protein.Results:1.Pathological changes of endometrium1.1HE staining:the morphology of endometrial epithelium in BSHX group and Progynova group was better than that of the model group.But BSHX group and Progynova group coundn’t reach the level of the normal group.The endometrial thickness and the number of glands in BSHX group and Progynova group were significantly higher than those of the model group,but lower than those of the normal group.There was no significant difference in endometrial thickness between the two groups(P>0.05).The number of glands in BSHX group was significantly more than that in Progynova group(P<0.05).1.1 Scanning electron microscope observation:blank group:pinodes was mature,villi located at the bottom of pinodes flatly.model group:The structure of microvilli was disordered and sparse,and pinodes developed slowly.BSHX group:The structure of microvilli is disordered and sparse,pinocytosis is sudden and slow,the structure of microvilli on pinodes attachment site was relatively complete,pinodes was almost mature.Progynova group:The structure of microvilli was not clear,and shape of pinodes was not full,with the poor degree of development.1.2 The number of embryo implantation sites:the number of implantation sites in BSHX group and Progynova group were significantly more than that in model group,but less than that in the normal group.Compared with the Progynova group,The number of embryo implantation sites in BSHX group was significantly more than that in Progynova group,P<0.05.3.Expression of ECM formation and degradation related factors:by immunohistochemistry,RT-PCR and Western blot,the relative expression levels of MMP9 and VEGF in BSHX group and Progynova group were up-regulated compared with the model group,P<0.05,but those expression levels was lower than that in the the normal group.Compared with Progynova group,the expression levels of MMP9 and VEGF in BSHX group were up-regulated,with statistical difference(P<0.05).Compared with the model group,the expression of TGF-β1 in BSHX group and Progynova group were down regulated,P<0.05.However,there was no significant difference in the expression of TGF-β1 between BSHX group and Progynova group(P>0.05).4.Expression of PI3K/Akt/Oct4 pathway related factors:by immunohistochemistry,RT-PCR,Western blot detection,BSHX group,the Progynova group compared with the model group,the relative expression levels of PI3K,Akt,Oct4 were up-regulated(P<0.05),but the expression levels were lower than the the normal group.The expression levels of PI3K,Akt and Oct4 in BSHX group were significantly higher than those in Progynova group(P<0.05).Conclusions:Bushen Huoxue Recipe can improve the pathological state of endometrium in IUAs rats,with reducing the deposition of extracellular matrix by influencing the related factors of ECM,repairing the injury endometrium by upregulating the related factors and protein expression in PI3K/Akt/Oct4 signal pathway. |
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