| Endometriosis(Endometriosis,EMs)is a common disease in women of childbearing age.Up to now,the pathogenesis is unclear,and the treatment is still difficult.EMs is similar to tumor in that it has dysfunction of mitochondria,imbalance of proliferation and apoptosis in ectopic endometrium,however,the mitochondrial regulatory mechanism involved in the occurrence and development of EMs is still unclear.CHCHD2 mitochondrial protein can regulate the mitochondrial function and apoptotic characteristics of various tumor cells,promote migration and lead to tumor progression.Whether CHCHD2 participates in the apoptosis and migration of EMs by regulating mitochondrial function remains to be further clarified.EMs belongs to the category of blood stasis syndrome in Chinese medicine,and Wenjing Decoction,a classic prescription from synopsis of the Golden Chamber,is the ancestral prescription of Zhongjing for regulating menstruation and is commonly used in the treatment of EMs.But at present,the research on the mechanism of Wenjing Decoction in the treatment of EMs is still insufficient,which has become a bottleneck restricting its application.Preliminary studies have shown that the effect of Wenjing Decoction on EMs is related to the change of mitochondrial structure of ectopic endometrium,but its molecular target of regulating mitochondrial function is not clear.In this study,we attempted to explore the role of CHCHD2 in the pathogenesis of EMs,especially the mechanism related to the mitochondrial function and apoptosis of endometrial stromal cells(ESCs),to explore the pathogenesis of EMs and possible therapeutic targets of EMs;on this basis,to explore whether the regulatory effect of Wenjing Decoction on the mitochondria of EMs is related to the down-regulation of CHCHD2 expression.This study may provide new ideas for the study of the pathogenesis of EMs and the diagnosis and treatment of TCM,and to provide a new basis for the scientific connotation of Wenjing Decoction in the treatment of EMs.Part one Localization and expression of CHCHD2 in eutopic and ectopic endometrium of patients with endometriosisObjective: To determine the differential expression of CHCHD2 in ectopic and eutopic endometrium.Methods: Ectopic endometrium,eutopic endometrium of patients with EMs and normal endometrium were collected,the CHCHD2 localization and expression in different endometrium were observed by immunohistochemistry,and the m RNA and protein expression of CHCHD2 were detected by quantitative real-time PCR(q RT-PCR)and western blot assay.Results:1.Localization and expression of CHCHD2 in eutopic endometrium(Eu),ectopic endometrium(Ec)of EMs and normal endometrium(NE)CHCHD2 protein was mainly expressed in cytoplasm in NE,Ec and Eu,while a small amount of CHCHD2 was expressed in nuclei;the expression of CHCHD2 was low in NE,CHCHD2 expression in Eu was significantly higher than that in NE(P<0.01),CHCHD2 expression in Ec was further significantly increased(P<0.01),indicating that CHCHD2 expression was significantly different in varied types of endometrium.2.The expression of CHCHD2 m RNA and protein in Eu,Ec and NEThe expression of CHCHD2 m RNA and protein in Eu and Ec was significantly higher than that in NE(P<0.01),CHCHD2 m RNA and protein expression in Ec group was significantly higher than that in Eu(P<0.01).Summary: The expression of CHCHD2 in ectopic and eutopic endometrium of EMs was significantly increased,suggesting that CHCHD2 may involved in the occurrence of EMs.Part two: Mechanism of CHCHD2 regulating the mitochondrial function of ectopic endometrial stromal cells and mitochondrial-mediated apoptosis in the development of endometriosisObjective: To explore the effects of CHCHD2 on mitochondrial function,mitochondrial-mediated apoptosis,proliferation and migration of ectopic endometrial stromal cells(ESCs),and to clarify the role of CHCHD2 in the development of EMs.Methods: The ectopic ESCs of EMs patients were primarily cultured,and the cell localization of CHCHD2 was observed by laser confocal microscope.The CHCHD2 sh RNA interference plasmid were constructed and transfected into ESCs,the transfection efficiency was verified by q RT-PCR and western blot assay;the cells were divided into Control group,sh-RNA negative control group(sh-NC)and sh-CHCHD2 group,the detection indexes were as follows:The morphology and function of mitochondria in ESCs: The mitochondrial membrane potential(MMP),the content of cytochrome C(Cyt C)in mitochondria,the opening of mitochondrial permeability transition pore(MPTP),the permeability of cell membrane and the fluorescence intensity of nuclei were detected by high content screening analysis(HCS).The morphology and structure of mitochondria were observed by transmission electron microscope(TEM).Expression of mitochondrial-mediated apoptosis pathway proteins: Bax,Bcl-2 and Cleaved Caspase-3 were detected by western blot assay.Proliferation,apoptosis and migration of ESCs: Cell proliferation was detected by MTT assay;cell migration was detected by transwell assay;and apoptosis was detected by Annexin V-FITC/PI double staining method.Results:1.Primary culture and identification of ESCsESCs were successfully isolated,the cells were spindle shaped or triangular,a few cells were polygonal or irregular,the cells have strong refraction and the proportion of mitotic cells were large,the purity and positive rate of the cells were higher than 95%.2.Localization of CHCHD2 of ESCsIn ESCs,CHCHD2 was mainly expressed in mitochondria of cytoplasm and co-localized with Mito Tracker of MMP dyes.3.Screening results of sh RNA interference plasmidCompared with Control group,the expression of CHCHD2 m RNA and protein in sh-NC group had no significant change(P>0.05),sh RNA three guarantees one plasmid screening results showed that sh RNA1 group had the most obvious m RNA and protein knockdown(P<0.01),which could be used as the interference plasmid in the following study.4.Detection of HCS multi-parameter mitochondrial function and apoptosisCompared with Control group,there were no significant changes in mitochondrial function and apoptosis parameters in sh-NC group(P>0.05).In sh-CHCHD2 transfected cells,the MMP was depolarized,the MPTP opening,Cyt C content,membrane permeability were increased significantly,in addition,nuclear pyknosis and chromatin agglutination were observed,nuclear fluorescence intensity was significantly increased and cell numbers were significantly decreased(P<0.05 or P<0.01),the results suggesting that the mitochondrial function was damaged in sh-CHCHD2 group,accompanied by apoptosis,indicating that CHCHD2 could regulate the mitochondrial function and apoptosis of ESCs.5.Ultrastructure of mitochondriaCompared with Control group,the integrity of mitochondrial membrane was destroyed in sh-CHCHD2 group,some cristae were blurred or even disappeared,a few mitochondria swelled obviously,and the outer membrane was ruptured,indicating that CHCHD2 may participate in maintaining the mitochondrial morphology of ESCs.6.Expression level of mitochondrial-mediated apoptosis pathway proteins Bax,Bcl-2 and Cleaved Caspase-3Compared with Control group,there was no significant change in protein levels in sh-NC group(P>0.05),the expression level of Bcl-2 protein was significantly decreased and the expression levels of Bax and Cleaved Caspase-3 protein were significantly increased in sh-CHCHD2 group(P<0.01).7.Detection of cell proliferation and apoptosis rateCompared with Control group,the cell proliferation and apoptosis rate of sh-NC group had no significant change(P>0.05),the OD value of sh-CHCHD2 group was significantly decreased at 24,48 and 72h(P<0.01),and had timedependent manner;the apoptosis rate of sh-CHCHD2 group was increased obviously(P<0.01),suggesting that CHCHD2 knockdown could promote ESCs proliferation and play a negative role in apoptosis regulation.8.Analysis of cell migration abilityTranswell assay showed that compared with Control group,the migration ability of sh-NC group had no significant change(P>0.05),and the migration ability of sh-CHCHD2 group was significantly decreased(P<0.01),it is suggested that CHCHD2 is a migration promoting factor of ESCs.Summary: Downregulated of CHCHD2 in EMs ectopic endometrial stromal cells could cause mitochondrial dysfunction,morphological damage,and then lead to inhibition of cell proliferation,decrease of migration ability,and aggravation of apoptosis,all these indicated that CHCHD2 may play an important role in the development of EMs,CHCHD2 might be the target of mitochondrial regulation of EMs and play a positive role in the pathogenesis of EMs.Part three: Mechanism of Wenjing Decoction regulating the expression of CHCHD2 to induce mitochondrial damage and apoptosis in endometriosis ratsObjective: To explore the effect of Wenjing Decoction on the expression of CHCHD2,mitochondrial function and apoptotic characteristics in ectopic lesions of EMs rats,and to preliminarily clarify the mechanism of Wenjing Decoction in the treatment of EMs.Methods: The rat model of EMs was established by autologous transplantation,and the model was detected by small animal ultrasound imaging system.According to the volume of ectopic endometrium,the rats were divided into Model group,Wenjing Decoction high dose(19.4 g/kg,WJT-H)group,medium dose(9.7g/kg,WJT-M)group,low dose(4.85g/kg,WJT-L)group and Sham operation group,with 15 rats in each group,and treated by gavage for 6weeks.The volume of ectopic lesion was measured by ultrasound imaging and caliper;the morphology of ectopic endometrium was observed by HE staining;the expression of CHCHD2 m RNA and protein level were detected by immunofluorescence staining,q RT-PCR and western blot analysis;the level of tissue MMP was detected by JC-1 probe;the ultrastructure of mitochondria in ectopic endometrium was observed by TEM;the expression of mitochondrialmediated apoptosis pathway proteins Bax,Bcl-2 and Cleaved Caspase-3 were detected by western blot analysis;and DNA damage was observed by TUNEL staining.Results:1.Observation and volume detection of ectopic lesionsUltrasound showed that the ectopic lesion was located under the abdominal wall,showing a hypoechoic area,sometimes with obvious blood flow,and the inflammatory fluid in the lesion was anechoic black area.Compared with the Model group,the volume of ectopic lesions in Wenjing Decoction groups were significantly decreased after 3 weeks of treatment(P<0.01),and further reduced after 6 weeks of treatment(P<0.01),indicating that Wenjing Decoction could significantly inhibit ectopic endometrial implantation in a dose and timedependent manner.According to the naked eye,the ectopic lesions showed a vesicle-like structure,containing light yellow or brown inflammatory fluid,and most of the lesions had obvious angiogenesis on the surface or adhered to the surrounding tissues.Compared with the Model group,the ectopic lesions in each dose of Wenjing Decoction groups were significantly atrophied,the volume was decreased,the fluid in the capsule was decreased,and even formed a flat flake structure(P<0.01).2.Ectopic endometrial morphologyThe ectopic endometrium in Model group showed a typical endometrial morphology;the ectopic endometrium of each dose of Wenjing Decoction group had obvious degenerative changes,which showed that the arrangement of glandular epithelial cells was disordered and damaged,the arrangement of stromal cells was loose,and the endometrial denudation was obvious.3.The expression levels of CHCHD2 m RNA and protein in ectopic lesionsCompared with the Sham group,the expression levels of CHCHD2 m RNA and protein in Model group were significantly increased(P<0.01);compared with the Model group,the expression levels of CHCHD2 m RNA and protein in all Wenjing Decoction groups were significantly decreased(P<0.05 or P<0.01),suggesting that Wenjing Decoction in the treatment of EMs might be related to the down-regulation of CHCHD2 expression in ectopic lesions.4.Detection of MMP levelCompared with the Sham group,MMP in ectopic endometrium of Model group was increased significantly(P<0.01);compared with the Model group,MMP in all Wenjing Decoction groups were decreased significantly(P<0.01),the result confirmed that the therapeutic effect of Wenjing Decoction on EMs might be related to the induction of mitochondrial dysfunction.5.Ultrastructural observation of mitochondriaThe morphology of mitochondria in Model group was similar to that of normal endometrium,but the number of mitochondria were increased obviously;mitochondria were damaged in different degrees in each treatment group: the number of mitochondria were decreased significantly,the mitochondria were swelled,the cristae blurred or disappeared,a few mitochondria outer membrane ruptured,and the vacuolar degeneration was obviously occured.6.Expression level of mitochondrial-mediated apoptosis pathway proteins Bax,Bcl-2 and Cleaved Caspase-3Compared with the Sham group,the expression levels of Bax and Cleaved Caspase-3 in ectopic lesions of Model group were significantly decreased,and the expression level of Bcl-2 was significantly increased(P<0.05 or P<0.01);compared with the Model group,the expression levels of Bax and Cleaved Caspase-3 in WJT-M and WJT-H groups were significantly increased(P<0.05 or P<0.01),and the expression level of Bcl-2 in each dose Wenjing Decoction group was significantly decreased(P<0.01),these results indicated that Wenjing Decoction could activate the mitochondrial-mediated apoptosis pathway.7.Detection of DNA damageCompared with the Sham group,the apoptosis rate of Model group was decreased(P>0.05);compared with the Model group,the apoptosis rate of ectopic endometrium in each dose group was significantly increased(P<0.05 or P<0.01).Summary: Wenjing Decoction could significantly inhibit the growth of ectopic lesions,and had a good therapeutic effect on EMs rats.The mechanism might be related to down-regulating the expression of CHCHD2 in ectopic lesions,resulting in the damage of mitochondrial function and the activation of mitochondrial-mediated apoptosis pathway in ectopic endometrial cells.Part four: Regulatory effect of Wenjing Decoction on endometrial stromal cells with CHCHD2 overexpressionObjective: To explore the regulatory effect of Wenjing Decoction on CHCHD2 overexpression ESCs,and to further clarify the relationship between Wenjing Decoction treatment of EMs and CHCHD2 regulation.Methods: The serum of Wenjing Decoction was prepared,and the effects of Wenjing Decoction on proliferation and apoptosis of ESCs were observed by MTT and Annexin V-FITC/PI double staining methods;the cell model of CHCHD2 overexpression ESCs was established,and the cells were divided into Control group,CHCHD2-OE group,CHCHD2-OE + 10% control serum group(CHCHD2-OE+KBXQ),CHCHD2-OE + 10% Wenjing Decoction serum group(CHCHD2-OE+WJTXQ).The expression of CHCHD2 and mitochondrial-mediated apoptosis pathway proteins Bax,Bcl-2 and Cleaved Caspase-3 were detected by western blot analysis,the mitochondrial function and apoptosis were measured by HCS assay(mitochondrial function: MMP,Cyt C content;apoptosis: membrane permeability,nuclear fluorescence intensity,nuclear size and cell numbers);cell migration ability was detected by transwell assay.Results:1.Proliferation and apoptosis of ESCsCompared with Control group,5% and 10% WJTXQ could significantly reduce the OD value of cells(P<0.01),and the inhibition rates were 22.0% and23.9% respectively;compared with the corresponding concentration KBXQ,the inhibition rates were 29.7% and 33.7%,respectively.5% and 10% WJTXQ could also increase the apoptosis rate significantly(P<0.01).2.Expression level of CHCHD2 and mitochondrial-mediated apoptosis pathway proteins Bax,Bcl-2 and Cleaved Caspase-3Compared with Control group,the levels of CHCHD2 and Bcl-2 in CHCHD2-OE group were significantly increased,while the levels of Bax and Cleaved Caspase-3 were significantly decreased(P<0.01);compared with the CHCHD2-OE group,the expression of these proteins in CHCHD2-OE+KBXQ group had no significant change(P>0.05),while the levels of CHCHD2 and Bcl-2 in CHCHD2-OE+WJTXQ group were significantly decreased,the levels of Bax and Cleaved Caspase-3 were significantly increased(P<0.01).3.Detection of mitochondrial function and apoptosisCompared with Control group,the level of MMP was increased,the level of Cyt C was decreased,the membrane integrity was high,the nuclear morphology was complete,the nuclear fluorescence intensity was low,and the cell numbers were increased in CHCHD2-OE group(P<0.05 or P<0.01);compared with CHCHD2-OE group,the KBXQ had no significant effect on the above indexes(P>0.05),and the WJTXQ could significantly affect the mitochondrial function: reducing MMP,promoting the release of mitochondrial Cyt C,it can also affected cell apoptosis: increasing cell membrane permeability,enhancing nuclear fluorescence intensity,promoting nuclear pyknosis,and reducing the number of cells(P<0.05 or P<0.01),indicated that Wenjing Decoction could regulate the mitochondrial function and induce apoptosis of ESCs.4.Analysis of cell migration abilityCompared with Control group,the number of migrating cells in the CHCHD2-OE group were significantly increased(P<0.01);compared with the CHCHD2-OE group,the KBXQ had no effect on the cell migration ability(P>0.05),while the WJTXQ could significantly reduce the number of migrating cells to the lower ventricle(P<0.01),suggesting that Wenjing Decoction could down regulate the expression of CHCHD2 and inhibit cell migration.Summary: Wenjing Decoction can down regulate the expression of CHCHD2 in vitro,and then regulate the function of mitochondria and mitochondrial-mediated apoptosis pathway related protein levels,inhibit the proliferation and migration of ESCs,and induce apoptosis.Conclusions:1.The abnormal expression of CHCHD2 in eutopic and ectopic endometrium might be closely related to the pathogenesis of EMs.2.CHCHD2 is an important regulatory factor in the development of EMs:CHCHD2 down-expression could cause mitochondrial dysfunction,inhibition proliferation and migration ability of ESCs,and aggravation mitochondrialmediated apoptosis.3.The therapeutic mechanism of Wenjing Decoction on EMs rats might be related to the down-regulation of CHCHD2 expression in ectopic lesions,which leads to mitochondrial dysfunction and activation of mitochondrial-mediated apoptosis pathway in ectopic endometrial cells.4.Wenjing Decoction could down regulate the CHCHD2 expression of ESCs,regulate the function of mitochondria and mitochondrial-mediated apoptosis pathway related protein levels,inhibit the proliferation and migration of ESCs,and induce apoptosis. |
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