| Background:Endometriosis(EMs)is one of the common diseases of reproductive endocrine disorders and a globally prevalent and difficult-to-treat disease.Modern medicine primarily adopts symptomatic therapies such as "pseudo-pregnancy" and "pseudo-menopause" to manage endometriosis,which can alleviate patients’ clinical symptoms.However,these approaches have certain limitations and violate women’s physiological state,making it difficult to tolerate for long periods.Another option is surgical treatment,but it often carries a high recurrence rate.Endometriosis ranks as the leading gynecological disease burden worldwide,and currently,it has been included in chronic disease management.Traditional Chinese medicine generally treats this disease based on the "blood stasis" theory.However,due to the insufficient understanding of the clinical features of endometriosis,specifically the relationship between clinical symptoms,the growth and regression of lesions,and menstrual cycles,clinical treatment has encountered difficulties.At present,the pathogenesis of this disease is not well-defined,the molecular mechanisms of traditional Chinese medicine diagnosis and treatment are not clear,and there is a lack of systematic clinical solutions.To improve clinical efficacy,it is urgently necessary to conduct in-depth research on the etiology and pathogenesis of endometriosis and establish new treatment methods.The theory of Chong and Ren is the core of traditional Chinese medicine gynecological theory.Led by Professor Liang Ruining,the team has proposed the "New Theory of Chong and Ren" with the core concepts of "the kidney as the root of Chong" and orderly perfusion of Chong Meridian qi and blood into the uterus".This theory elucidates that kidney Qi is the driving force behind the perfusion of Qi and blood in the Chong pulse to the uterus.It reveals the one-way flow pattern of Chongmai Qi and blood into the uterus and clarifies that the sealing and absorption of kidney Qi are crucial for maintaining the "visceral" properties of the uterus.It innovatively proposes that "disorder in the flow of Qi in the Chongmai leads to stasis of blood deviating from its normal course," which is the key pathological mechanism of endometriosis.This theory scientifically explains the relationship between this disease and the menstrual cycles.1."Disorder" is manifested by pain,mass(lumps),menstrual irregularities and infertility as main features.The clinical symptoms and development of the disease are closely related to the uterus and menstrual cycles.Additionally,endometriosis is often complicated by hyperprolactinemia,which is related to the counterflow of Qi in the Chong vessel,preventing the flow of Qi and blood to the uterus and causing its upward transformation into milk.2.The causes of "Reversal" include kidney deficiency,which leads to Qi of the Chong Meridian rushing through in a reversed way;insufficient yang of the heart,which activates the Yin Evil and in turn leads to Qi reversal of the Chong Meridian;Rampant Liver depression,leading to Qi-Blood reversal of the Chong Meridian;Qi deficiency of Yang Ming Meridian,leading to stomach Qi reversal.Based on this new pathogenesis,the "calming chong,lowering reversal,resolving stasis and unblocking collaterals" method was adopted,and the "Ping-Chong-Jiang-Ni formula" was used to treat endometriosis,the clinical efficacy of which has been verified.Under the guidance of the new pathogenesis theory,in-depth research was conducted on the main active ingredient of Guizhi(Cinnamon twig),cinnamaldehyde(CA),to successfully screen out cinnamaldehyde as a potential drug for endometriosis.The main research objectives are to study the effects of CA on endometriotic stromal cells through in vivo and in vitro analysis and explore its possible molecular mechanisms,providing potential new strategies for the development of therapeutic drugs for endometriosis.Main research methods:1.Isolate and purifiy human primary ectopic endometrial stromal cells(EESCs),and identify cells by immunocytochemistry;screen the optimum concentration of CA intervention on EESCs by CCK8 method;randomly divide the in vitro cultured EESCs into CONT group,DMSO group and CA group.Transwell chamber assay was used to evaluate the effects of CA intervention on EESCs migration and invasion.2.4D-DIA proteomics technology was used to analyze the differentially expressed protein spectrum in CA-treated EESCs.3.Establish EMs rat models by autologous transplantation,The rats were divided into sham operation group,model group,low,middle and high dose CA groups and progesterone group.The sham operation group and model group were given distilled water by gastric lavage,the low,middle and high dose CA groups were given 25 mg/kg,50 mg/kg and 100 mg/kg by gavage respectively,once a day for continuous treatment for 3 weeks.The weight of ectopic endometrial tissue from each group of rats was measured,Hematoxylin-eosin staining was used to observe histopathological changes in rat endometrial tissues,and immunohistochemistry was used to verify the results of proteomics analysis and observe the expression level of heme oxygenase-1(HMOX-1)protein in rat lesional endometrial tissue.4.The 12 Z endometriosis cell line was selected,DMSO group and CA group were set up,and Western Blotting was used to verify the expression of HMOX-1 after CA intervention;After siRNA transfection,the DMSO group,CA group,CA + si-NC group and CA + si-Mix group were set up,and Western Blotting was used to detect the effect of siRNA transfection on CA intervention on HMOX-1 expression.Transwell chamber assay was used to detect the effects of siRNA transfection on CA intervention on EESCs migration and invasion.Results:1.Immunocytochemistry results showed positive staining for vimentin and negative staining for cytokeratin,with PBS control showing negative staining.This suggests that the purity of the isolated and cultured EESCs reached 90%-95%.CCK-8 assay results demonstrated that CA exhibited concentration-dependent inhibition of cell viability within 24 hours.The IC50 value of CA at 100 μM for 24 hours was determined to be 44.74 μM,indicating that 100 μM was the optimal intervention concentration of CA.Therefore,100 μM was chosen for subsequent experiments.Transwell chamber assay results showed that the migration and invasion of EESCs in the CA group were significantly reduced compared to the control group(P<0.05),indicating that CA had inhibitory effects on the migration and invasion of EESCs.2.Proteomic analysis revealed a total of 5062 identified proteins in both the CA intervention and control groups,with 4236 proteins in common between the two groups.Among them,there were 214 proteins with expression differences greater than 1.5 times(P<0.05),including 57 upregulated and 157 downregulated proteins.Through comprehensive analysis,HMOX-1 was selected as a potential target protein for CA intervention in EESCs.3.In the EMs rat model,the weighing of ectopic endometrial tissues in each group showed that the model group had the highest tissue weight(n=6,P<0.01).The middle-dose group had significantly lower tissue weight compared to the low-dose group,high-dose group,and progesterone group(P<0.05),indicating that CA intervention effectively reduced the size of ectopic endometrial lesions in EMs rats,with the middle-dose group showing the best effect.Histopathological examination revealed that in the model group,the normal in situ endometrial tissue had high columnar epithelial cells arranged tightly,well-developed glands and stromal cells,while the ectopic endometrial tissue had a higher degree of inflammatory infiltration and poorer cell morphology.Compared to the model group,the treatment groups showed improvement in the degree of inflammatory infiltration and cell morphology in the ectopic endometrial tissue,indicating that CA could alleviate inflammation in the EMs rat model.Immunohistochemical results showed that compared to the sham operation group,the expression level of HMOX-1 in the ectopic endometrial tissue of the model group was significantly decreased(P<0.05),while compared to the model group,the expression level of HMOX-1 was significantly increased in each treatment group(P<0.05),suggesting that CA intervention could increase the expression level of HMOX-1 in the ectopic endometrial tissue of rats.4.In the 12 Z cells line of endometriosis,Western Blotting experiment showed that compared with DMSO group,CA intervention significantly increased the expression of HMOX-1 protein in cells(P<0.05),which was consistent with proteomics analysis;The RT-q PCR experiment results showed that the expression of HMOX-1 in the siRNA-Mix group was lower than that in the NC group(P<0.05),indicating that the transfection was effective and the cells could be used for subsequent experiments.After transfection,Western Blotting results showed that compared with the CA group,the expression of HMOX-1 protein in the CA+si-Mix group was significantly downregulated(P<0.01),confirming that CA intervention was effective and activated the expression of HMOX-1 in 12 Z cells to a certain extent.The Transwell cell results showed that compared with the DMSO group,the migration and invasion ability of the CA group was significantly downregulated(P<0.01),confirming that CA activated HMOX-1expression in 12 Z cells.Compared with siRNA-NC group,HMOX-1 silencing could promote12 Z cells migration and invasion to some extent(P<0.05).Compared with the si-Mix group,the cell migration and invasion ability of CA+si-Mix group were significantly down regulated(P<0.05),suggesting that the inhibition of CA on cell migration and invasion might be antagonistic to the silencing of HMOX-1.Therefore,HMOX-1 is a potential molecular target for CA treatment of EMs.Conclusion:In summary,CA can effectively inhibit the migration and invasion of endometrial stromal cells and inhibit ectopic lesions in EMs rats.Proteomics and molecular mechanism studies confirmed that the up regulation of HMOX-1 expression mediates the effect of CA on inhibiting the migration and invasion of endometrial stromal cell migration. |
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