Mechanism Study Of Rhubarb-Peach Kernel On Regulating Mitochondrial Homeostasis In Adenomyosis

Background Adenomyosis(AM)is a common gynecological disease that is characterized by gradually increasing dysmenorrhea and abnormal menstrual volume.It is also a known cause of secondary infertility in women.AM is caused by the endometrium abnormally infiltrating and growing into the myometrium.At present,clinical conservative treatments include hormone drugs and non-steroidal anti-inflammatory drugs,but these options come with drawbacks such as ovulation inhibition,liver and kidney function damage,poor efficacy,and high recurrence rate.In recent years,Traditional Chinese Medicine(TCM)has gained more attention due to its unique advantages in significantly improving the clinical symptoms and pathological infiltration of AM without interfering with normal endocrine function.The occurrence of AM is the result of hypoxic damage to the eutopic intima.Mitochondria are the most advanced intracellular reactive sensing system and are extremely sensitive to hypoxic damage.Mitochondrial damage leading to oxidative stress is an important mechanism and potential therapeutic target for AM.Traditional Chinese medicine,particularly for removing blood stasis,has been proven to regulate the level of mitophagy,activities of mitochondrial respiratory chain enzymes,antioxidant enzymes,improve the stability of mitochondrial membrane,repair damaged mitochondria,and inhibit the abnormal proliferation of cells in various diseases under hypoxic microenvironments.However,there have been no relevant studies conducted on AM.As such,this study aims to explore the potential therapeutic mechanism of RP on AM by targeting mitochondrial homeostasis in the endometrium.Objective To explore the regulatory mechanism of RP on endometrial mitochondrial homeostasis in adenomyosisi(AM).Methods(1)Collect clinical specimens and use Western Blot to compare the expression levels of mitochondrial autophagy proteins in the endometrium between patients with adenomyosis(AM)and the control group.(2)Human endometriotic ectopic endometrial cells(hEM15A)were cultured in vitro and immortalized.CCK-8 assays were performed to test the proliferation rate of hEM15 A cells before and after treatment with RP.The ROS changes in hEM15 A cells were labeled with DCFH-DA fluorescent probe.Mitochondrial membrane potential was observed with JC-1 fluorescent probe.Antioxidant enzyme activities of hEM15 A cells were detected by using malondialdehyde(MDA)assay,superoxide dismutase(SOD)assay,and glutathione peroxidase(GSH-PX)assay.Mitochondrial autophagosome was labeled with fluorescent probe.Scratch tests and Transwell experiments were conducted to compare the migration and invasion capacities of cells before and after RP treatment.Western Blot was used to detect the expression levels of PINK1,Parkin,optineurin(OPTIN),nuclear dot protein 52(NDP52),and P62 proteins that are related to mitochondrial autophagy in hEM15 A cells.(3)8-week-old ICR female mice were randomly divided into control group(n=8),model group(n=11),and Rhubarb-Peach Kernel(RP)group(n=14).A pituitary transplantation method was used to construct the AM model,with RP group mice orally administered RP decoction(0.3125g/Kg/day),while control group and model group mice were orally administered an equivalent amount of physiological saline for a total of 8weeks.The pathological infiltration of the uterus was detected by HE staining;the ultrastructural changes of endometrial cells were observed by Transmission Electron Microscope(TEM);the expression and localization of PTEN-induced putative kinase 1(PINK1)/Parkinson protein 2(Parkin 2)protein induced by the PTEN signaling pathway of mitochondrial autophagy were detected by immunofluorescence.The level of mitochondrial reactive oxygen species(ROS)in the mouse uterus was detected by DCFH-DA fluorescence probe.Results(1)Clinical specimen: Western Blot results showed that compared with the normal control group,the expression levels of mitochondrial autophagy-related proteins PINK1,Parkin,P62,and OPTIN in the endometrium of the AM group were significantly increased.(2)In vitro experiment 1)The CCK-8 test outcomes indicated that RP exhibited a dose-dependent inhibition on the abnormal proliferation of hEM15 A cells.2)The findings revealed that treatment with RP improved the mitochondrial membrane potential of hEM15 A cells,and significantly reduced their ROS levels(P < 0.01).3)Furthermore,RP treatment resulted in a notable increase in the activities of key mitochondrial antioxidant enzymes,including Cu-Zn superoxide dismutase(SOD),Mn-SOD,and glutathione peroxidase(GSH-Px)in hEM15 A cells(P < 0.01,0.001).Additionally,the level of malondialdehyde(MDA)was significantly reduced(P < 0.01).4)Observations indicated a robust correlation between mitochondria and lysosomes in hEM15 A cells,where the number of instances of co-localization decreased significantly following RP treatment(P < 0.05).5)According to the findings from both Scratch and Transwell experiments,it was observed that RP treatment had a substantial inhibitory effect on the migration and invasion ability of hEM15 A cells.6)After RP treatment,the Western Blot results demonstrated notable downregulation of mitophagy protein PINK1,along with Parkin and its subsequent proteins,OPTIN,NDP52,and P62 in hEM15 A cells(P < 0.01,0.001).(3)In vivo experiments: 1)The pathological examination results indicated that in the modeled tissues,the endometrial cells infiltrated into the myometrial layer.However,following RP treatment,there was a noticeable decrement in the degree of infiltration.2)The TEM analysis revealed that the mitochondria in both the endometrial epithelial cells and stromal cells were swollen in the model group,with mitochondrial cristae disappearing and mitophagosomes formation occurring.However,after RP treatment,there was an increase in the number of mitochondria observed in both endometrial epithelial and stromal cells.In addition,no significant swelling of mitochondria was observed,and clear cristae were visible.3)Based on the immunofluorescence results,both PINK1 and Parkin were found to be expressed in the stromal cells and glandular epithelial cells of the endometrial layer.When compared with the control group,a significant up-regulation in the expression of both PINK1 and Parkin was observed in the model group(P < 0.001).However,following RP treatment,there was a significant decrease noted in their expression(P < 0.05,0.001).Conclusion RP can regulate mitochondrial homeostasis in AM endometrial cells through multiple pathways,such as up-regulating the activities of mitochondrial antioxidant enzymes,inhibiting mitochondrial autophagy,and improving mitochondrial membrane potential,thereby reducing oxidative stress injury.