| The progression of ectopic lesions is the key problem leading to the development of Endometriosis and the high recurrence rate.Depression and Endometriosis promote each other,but the specific mechanism has not been reported.Depression,as a stressor,promotes the secretion of norepinephrine from the SympatheticoAdrenomedullary system,activates regulatory T cells,inhibits the function of effector T lymphocytes,impedes the clearance of lesions,and promotes the progression of tumors and other diseases.Our study aims to explore the role of β2-AR/Treg signaling pathway in the progression of ectopic lesions in Endometriosis patients with depression,and elaborate mechanism of Huoxue Xiaoyi Decoction,providing new ideas for Traditional Chinese Medicine treatment and long-term management of Endometriosis.Objective(1)To analyze distribution and transformation characteristics of syndrome types of Endometriosis patients.(2)To investigate role of depression-mediated immunosuppression induced by regulatory T cells in the progression of Endometriosis.(3)To explore the mechanism of Huoxue Xiaoyi Decoction in improving depression and controlling the progression of ectopic lesions in Endometriosis.MethodsIn clinical trial ⅠWe summarized syndrome types of 410 patients at the first visit and 261 subsequent visit patients every 3 months in Endometriosis.The effects of age,body mass index,disease course,menstrual volume,dysmenorrhea score,lesion type,lesion size,surgical history,and Chinese medicine course on syndrome type were analyzed.In clinical trial ⅡThirty Endometriosis patients of qi stagnation and blood stasis syndrome combined with depression were included as the case group and ten healthy women as control.The distribution characteristics of peripheral neurotransmitters and T lymphocytes were analyzed.In vivo experiment ⅠTwenty-five SPF female SD rats aged 8 weeks were selected and divided into blank group(Control),Sham group(Sham),EM group(EM),EM depression group(EM+CUMS)and depression group(CUMS),with 5 rats in each group.Chronic unpredictable mild stress was used to establish depression model and allogeneic endometrial transplantation was used to establish Endometriosis model.Weighing,sugar water preference test and open field test were carried out at 6 weeks after modeling.The volume of ectopic lesions was measured.Morphology of rat hippocampus was observed by Nissl’s staining.The content of NE,DA and 5-HT in peripheral blood of rats was detected by ELISA.The proportion of Treg,CTLA-4 and CD3+CD8+T cells in ectopic lesions was detected by flow cytometry.Western blot was used to detect BDNF proteins in hippocampus and cAMP,P-CREB and P-PKA proteins in ectopic lesions.Genes including cAMP,CREB and PKA in ectopic lesions was detected by qRT-PCR.Immunohistochemistry was used to detect the content ofβ2-AR,IL-2,perforin and granzyme in ectopic lesions.In vivo experiment ⅡTwenty SPF female SD rats aged 8 weeks were selected.They were divided into blank group(Control),EM depression group(EM+CUMS),Huoxue Xiaoyi Decoction low-dose group(HXXYF-L)and Huoxue Xiaoyi Decoction high-dose group(HXXYF-H),with 5 rats in each group.EM+CUMS group,HXXYF-L group and HXXYF-H group all underwent depression modeling and Endometriosis modeling.Endometriosis modeling was performed on day 14 of depression modeling.On day 7 of Endometriosis modeling,HXXYF-L group and HXXYF-H group were given low-dose and high-dose Huoxue Xiaoyi Decoction respectively,for a total of 3 weeks.Control group and EM+CUMS group were given saline.Weighing,sugar water preference test and open field test were carried out at 6 weeks after modeling.Samples were collected and tested.ResultsIn clinical trial Ⅰ(1)Qi stagnation and blood stasis syndrome was the main syndrome type of Endometriosis,accounting for 62.9%,followed by qi deficiency and blood stasis syndrome.(2)After taking Chinese medicine,syndrome types were transformed,and the proportion of qi stagnation and blood stasis syndrome was significantly increased,up to 84.3%.Three and nine months of medication were the key points of syndrome type transformation.(3)The course of Chinese medicine has significant correlation with syndrome type transformation,which is an important factor to promote syndrome type transformation.In clinical trial Ⅱ(1)Compared with healthy women,the level of norepinephrine in peripheral blood of EM patients with depression was significantly higher(P<0.05).(2)Compared with normal reference range,the proportion of CD3+T cells,CD4+T cells,CD8+T cells in peripheral blood of EM patients with depression was roughly normal.The abnormal rate of regulatory T cells was 83.33%,which were all higher.(3)Compared with healthy women,there were no significant differences in the proportion of CD3+T cells,CD4+T cells and CD8+T cells in peripheral blood of EM patients with depression(P>0.05).(4)Compared with healthy women,proportion of regulatory T cells in peripheral blood of EM patients with depression was significantly higher(P<0.01),and the highest value was 14.55%,which was about twice the upper limit of the normal range.There were no significant differences of CD4+CD25+T cells compared with healthy women(P>0.05).(5)Compared with healthy women,there were no significant differences in the proportion of CD8+CD28-T cells and CD8+CD28+T cells in peripheral blood of EM patients with depression(P>0.05).In vivo experiment Ⅰ(1)Compared with Control group,weight of EM group,EM+CUMS group and CUMS group was significantly decreased(P<0.001).(2)Compared with Control group,sugar water consumption was significantly reduced in EM+CUMS group and CUMS group(P<0.001).Compared with EM group,consumption rates of sugar water were significantly lower in EM+CUMS group and CUMS group(P<0.001).(3)Compared with Control group,total exercise distance,total exercise time and central time in CUMS group were significantly decreased,while total rest time was significantly increased(P<0.05).The changes of total exercise distance,total exercise time and total rest time in EM+CUMS group were more significant(P<0.01),and central time was significantly decreased(P<0.05).There was no significant difference between EM group and other groups in the experiment trajectory of open field test.(4)In EM+CUMS group and CUMS group,hippocampal cells were significantly reduced,with irregular cell morphology,enlarged space,disordered arrangement,disappearance of nucleus,and expression level of BDNF protein was significantly decreased.Compared with Control group,peripheral blood NE was significantly increased,DA and 5-HT were significantly decreased in EM+CUMS group and CUMS group.Compared with Control group,the content of NE,DA and 5-HT in peripheral blood of EM group was changed to some extent,and expression level of BDNF protein in hippocampus was decreased(P>0.05).(5)Compared with EM group,volume of ectopic lesions in EM+CUMS group was significantly increased(P<0.01),the proportion of regulatory T cells in ectopic lesions was significantly increased(P<0.05),and CTLA-4 on the regulatory T cells was highly expressed(P<0.05).(6)Compared with EM group,proportion of cytotoxic T lymphocytes in ectopic lesions of EM+CUMS group was lower(P<0.01),and content of IL-2,perforin and granzyme was significantly decreased,with statistically significant difference.(7)Compared with EM group,β2-AR expression was significantly increased in ectopic lesions of EM+CUMS group(P<0.05),and content of cAMP,P-CREB and P-PKA protein was significantly increased,with statistical significance.The mRNA expression level of cAMP,CREB and PKA in EM+CUMS group was significantly increased(P<0.001).In vivo experiment Ⅱ(1)Compared with EM+CUMS group,weight of HXXYF-L group and HXXYF-H group was significantly increased(P<0.001).(2)Compared with EM+CUMS group,consumption rate of sugar water in HXXYF-L group and HXXYF-H group was significantly increased(P<0.001),and there was no significant difference with that in Control group.(3)Compared with EM+CUMS group,total exercise distance and total exercise time of HXXYF-L group and HXXYF-H group were significantly increased,while total rest time was decreased(P<0.001).Central time of HXXYF-L group was increased compared with EM+CUMS group(P<0.05).There was no significant difference in behavioral test between the two groups and Control group(P>0.05).(4)Compared with EM+CUMS group,number of hippocampal cells HXXYF-L group and HXXYF-H group increased,the arrangement was more orderly and the morphology was more complete.The expression level of BDNF in hippocampus of HXXYF-L group was significantly higher than EM+CUMS group(P<0.01).NE in peripheral blood of rats in HXXYF-L group and HXXYF-H group was significantly decreased,and content of DA and 5-HT was significantly increased,with statistically significant difference.(5)Compared with EM+CUMS group,volume of ectopic lesions in HXXYF-L group and HXXYF-H group was significantly reduced(P<0.01),proportion of regulatory T cells in ectopic lesions was significantly reduced(P<0.01),and CTLA-4 expression on regulatory T cells was significantly down-regulated(P<0.01).(6)Compared with EM+CUMS group,proportion of cytotoxic T lymphocytes in ectopic lesions in HXXYF-L group and HXXYF-H group was significantly increased(P<0.05),and content of IL-2,perforin and granzyme was significantly increased(P<0.01).(7)Compared with EM+CUMS group,both low dose and high dose Huoxue Xiaoyi Decoction could effectively reduce expression of β2-AR in ectopic lesions,and the difference was statistically significant.The protein content of cAMP,P-CREB and P-PKA in ectopic lesions in HXXYF-L group and HXXYF-H group was decreased to different degrees with statistically significant difference.The mRNA expression level of cAMP,CREB and PKA in ectopic lesions was significantly decreased in both groups(P<0.001).Conclusion(1)Qi stagnation and blood stasis is the essence of Endometriosis,throughout the whole process of disease development.(2)Depression may promote EM progression by activating the SympatheticoAdrenomedullary system to release norepinephrine and regulate regulatory T cell-mediated immunosuppressive microenvironment in ectopic lesions.(3)Huoxue Xiaoyi Decoction can improve depression,regulate β2-AR/Treg signaling pathway,enhance the ability of killing ectopic lesions,and control the progression of ectopic lesions. |
PDF Full Text Request