| ObjectiveThe pathogenesis of endometriosis(EMS)has not been fully elucidated.Luoshi Endometriosis Formula has been used in clinicalpractice for many years with satisfactory effects,but there are few studies on its mechanism.This study intends to analyze the related factors of EMS through clinical study,and in vitro experimental study will study the mechanism of action of Luoshi Endometriosis Formula from the cellular level.Ovarian EMS has a highc-linical incidence and is convenient to obtain materials.It is the first choice for primary culture experiments of ectopicendometrial cells in vitro.EMS has carcinoid biological behavior.We chose autophagy,which plays an important role in the develop-ment of both cancer and EMS,as our starting point.In this study,Temsirolimus,an mTOR inhibitor,was used as the control drug to observe how Luoshi Endometriosis Formula regulates PI3K/Akt/mTORsignaling pathway and autophagy mechanism.Results1.Clinical researchThe relevant information of 105 patients with EMS who under-went surgical treatment in the First Affiliated Hospital of Gua-ngzhou University of Chinese Medicine was collected.All patientswere pathologically diagnosed as EMS after surgery.The question-naire was designed to collect the general information of the patients,the surgical data and the syndromes data of four diagnoses of Chinese medicine,such as looking,smelling,asking,and feel-ing the pulse,and to analyze the related factors and the distri-bution of Chinese medicine syndromes.2.In vitro experimental researchCollected 20 cases of ectopic endometrial tissue of ovarian EMS patients and normal endometrial tissue of patients with inf-ertility due to tubal obstruction who underwent surgery in the First Affiliated Hospital of Guangzhou University of Chinese Me-dicine,and extracted primary cells from them for culture and pa-ssage.Tissue sources are divided intoe ctopic endometrial cells(Oc group)and normal endometrial cells(Cc group).Results1.Clinical research(1)Proportion distribution of EMS related factors:(1)Age and Region:EMS patients were mainly in the range of 26-35years old,accounting for 49.53%;71.43% in Guangdong province.(2)BMI:68.57%(72/105 cases)were within the standard range,16.19%(17/105 cases)were underweight,15.24%(16/105 cases)were overweight and obese.(3)Birth situation:Among the 105 patients,45.71%(48/105)had 1 to 2times of pregnancy,18.1%(19/105)had more than 3 times ofpregnancy;43.18%(46/105)had 1 to 2 times of delivery,3.81%(4/105)had more than 3 times of delivery;33.33%(35/105)had 1 to 2 times of abortion,and 4.76%(5/105)had more than 3 times ofabortion.36.19%(38/105 cases)had never been pregnant,and 20%(21/105 cases)had fertility requirement but had never been pre-gnant.(4)Tumor related indicators: in all patients,CA125 elevationaccounted for 64.86%(48/74 cases),CA199 elevation accounted for 36.23%(25/65cases),31.75%(20/63 cases)of both increased.(5)rAFS stage:in all patients,mainly in the stage Ⅲ and Ⅳ,were 36.19%(38/105 cases),Ⅰperiod accounted for 20%(21/105cases),Ⅱperiod accounted for 7.62%(8/105 cases).(6)Menstruation situation: irregular cycle accounted for 19.1%(19/105cases);less menstrual volume was 15.24%(16/105 cases),and more menstrual volume was 7.62%(8/105 cases).Menstrual colorwas dark red in39.05%(41/105 cases)and dull in 12.38%(13/105 cases).Blood clots in menstruation accounted for 66.67%(70/105 cases),accompanied by abdominal pain accounted for 54.29%(57/105 cases),waist acid accounted for47.62%(50/105 cases),breast painaccounted for 48.57%(51/105 cases).(2)The proportion distribution of Chinese Medicine syndromes: this study found that in EMS patients,the syndromes of phlegm and blood stasis accounted for 25.71%(27/105 cases),the syndromes of cold coagulation and blood stasis accounted for 18.1%(19/105 cases),the syndromes of qi deficiency and blood stasis accounted for 17.14%(18/105 cases),the syndromes of qi stagnat-ionand blood stasis accounted for 14.29%(15/105cases),and the syndromes of kidney deficiency and blood stasis and wet blood stasis accounted for 12.38%(13/105 cases).2.In vitro experimental research(1)Cell culture:The success rates of separation and culture of ectopic endometrial cells and normal endometrial cells were 85%(17/20 cases)and 75%(12/20 cases),respectively.Both types ofcells can adhere to the wall within 24 hours.It takes about 10-12 days for ectopic endometrial cells and eutopic endometrial cells to grow and fuse to more than 90%,andabout 8-10 days fornormal endometrial cells.(2)Cell identification:Cell slides were made from cells passed through the third generation.Immunocytochemical identification was performed with anti-Vimentin Antibody and anti-PCK-26 Antibody,showing brownyellow cytoplasm changes.(3)Drug concentration screening:CCK-8 method was used to detect cell proliferation to determine drug concentration.Finally,Luoshi Endometriosis Formula was selected as 20mg/ml,and Temsi-rolimus concentration was 0.001 m M.(4)Detection of related protein expression:Wester Blot methodwas used to detect the expression of LC3Ⅰ,LC3Ⅱ,PI3 K,Akt,4EBP-1,S6K1 and mTOR protein in each group.The results indicated that in the Cc group,compared with the blank group,the protein level of LC3Ⅰ and LC3Ⅱ in the group increased,and only the protein level of LC3Ⅰ increased in the Luoshi Endometriosis Formula group,only LC3Ⅰ protein increased at P<0.05.The prot-ein level of PI3 K,4EBP1,S6K1,mTOR and Akt was decreased.The protein level of PI3 K,4EBP1,S6K1 were all decreased by P<0.05.The protein level of LC3Ⅰ and LC3Ⅱ in Temsirolimus group was increased.The elevated levels of both proteins were P<0.05.The protein level of PI3 K,4EBP1,S6K1,mTOR and Akt was decreased.The protein level of PI3 K,4EBP1 and mTOR decreased P<0.05.In the Oc group,compared with the blank group,the protein level of LC3Ⅰ and LC3Ⅱ in the Luoshi Endometriosis Formula group was increased,all of which were P<0.05.The protein levels of PI3 K,Akt,4EBP1,S6K1 and mTOR decreased,all with P<0.05.In the Tems-irolimus group,the protein level of LC3 was increased(P<0.05),and the protein level of PI3 K,Akt,4EBP1,S6K1 and mTOR was decreased(P<0.05).Conclusion1.It was found in the survey that EMS patients were mainly in the range of 26-35 years old,and the CA125 elevation accounted for 64.86%.CA199 elevation accounted for 36.23%.2.The syndromes of Chinese medicine are mainly the syndrome of mutual association of phlegm and blood stasis and the syndrome of cold coagulation and blood stasis,followed by qi deficiency and blood stasis syndrome of qi stagnation and blood stasis syndrome of kidney deficiency and blood stasis syndrome of wet blood stasis syndrome.3.Luoshi Endometriosis Formula may enhance autophagy by inhibiting the activation of PI3K/Akt/mTOR signaling pathway to play a therapeutic effect on ovarian EMS. |
PDF Full Text Request