| Objective:Endometriosis is difficult to cure and the high recurrence rate has always been a thorny problem in gynecology.In this study,the empirical prescription Juantong Decoction was used as the research object,and the rat autotransplantation method was used to establish the EMs model to study the molecular mechanism of Juantong Decoction regulating the endoplasmic reticulum stress-activated PERK/eIF2α pathway to mediate ectopic endometrial autophagy.To clarify the mechanism of endoplasmic reticulum-mediated PERK/eIF2α pathway in regulating endometriotic endometrial autophagy and the intervention mechanism of Juantong Decoction on it,and to explore the biological nature of the pathogenesis of EMs from the molecular level,and to provide for Juantong Decoction Provide theoretical basis for the clinical application.Methods:70 SD rats were randomly divided into sham operation group,model group,Juantong Decoction(high,medium,and low)dose group,gestrinone group and ERS group.Except for sham operation group,all other groups were replicated into EMs Rat model,28 days after modeling,the status of EMs modeling was detected,continuous gastric administration for 28 days,measurement and evaluation of ectopic lesion volume and pelvic adhesion degree before and after medication;detection of serum CRP,E2 content of each group of rats;and in light microscope Observe the pathological changes of the ectopic endometrium of rats in each group;immunohistochemistry and real-time fluorescent quantitative PCR were used to detect the protein and gene expression of PERK,eIF2α,and LC3B.Results:(1)General conditions of rats:The rats were fed normally before the operation,without special conditions.In the first 3 days after the operation,the rats in the sham-operated group had reduced activities and food intake,slightly increased drinking water,messy and dim hair,pale pink nails,and dark purplish red tongue,and recovered as usual 3 days after the operation.Model rats have dry hair within one week after operation,eat less food and move less,curl up hair,lose energy,drink more water,and lose weight than before;some rats have slow growth at the surgical mouth,abdominal bulging,and no bowel movements.After treatment with penicillin,Some rats can resume defecation,and recover as usual one week after the operation.During the experiment,3 model rats died due to infection and intestinal obstruction.(2)Rat pelvic adhesion score:Compared with the sham operation group,the degree of pelvic adhesion in EMs rats was severe and the difference was significant(P<0.001);after the intervention of medication,compared with the model group,the high and medium doses of Juantong Decoction and pregnant women Pelvic adhesions in the trienone group were significantly reduced(P<0.01),the difference was the most significant in the high-dose group(P<0.001),and there was no significant difference in the low-dose Juantong Decoction group and the ERS group(P>0.05);the traditional Chinese medicine group was compared in pairs No significant difference(P>0.05).(3)Comparison of the volume of ectopic lesions in rats:After the intervention.the ectopic lesions in the Juantong Decoction group were significantly smaller than before the intervention,and the difference was significant,(P<0.01).The ectopic volume of the gestrinone and ERS groups was higher than before.There was no significant difference(P>0.05);compared with the model group,the high,medium,and low-dose Juantong Decoction groups and the ERS group were statistically significant(P<0.05),and the high-dose group had significant differences(P<0.01);There was no difference in the pairwise comparison between the high,medium,and low-dose Juantong Decoction groups(P<0.05);compared with the model group,the inhibition rates of the Juantong Decoction and gestrinone groups were statistically significant(P<0.05).(4)The pathological changes of the rats in each group were detected by HE:the endometrium of the rats in the sham operation group was continuous,and the morphology of glands and interstitial cells were regular,and the structure level was clear;the low-dose of Juantong Decoction and the endometrium of the gestrinone group The epithelium is tall columnar,with poor continuity,most of the cells have subnuclear vacuoles,and vascular distribution can be seen;the intima thickness of the high,medium dose,gestrinone and ERS groups is different,and the epithelium falls off and appears petal-like.The number of glands decreased continuously,and the interstitial layer became thinner,which was obvious in the high-dose group.(5)ELISA detection of rat serum CRP and E2 content:Compared with the sham operation group,the CRP and E2 content of rats in the EMs group decreased with significant difference(P<0.001);compared with the model group,the high,medium and high doses of Juantong Decoction The levels of CRP and E2 in the gestrinone group were significantly decreased,both of which had a significant difference(P<0.01).There was no significant difference in CRP between the low-dose Juantong Decoction group and the ERS group((P>0.05).The serum E2 of the ERS group was The content decreased significantly,and the difference was significant(P<0.0001).(6)Immunohistochemical detection of PERK,eIF2α,LC3B protein location and content:PERK,eIF2α are mainly located in the cytoplasm,nucleus and glandular epithelium of the stromal layer,and the LC3B protein is mainly located in the glandular epithelial cells and stromal cells of the ectopic endometrium.In the cytoplasm.The PERK,eIF2α,and.LC3B protein expressions in EMs model rats were low,compared with the sham operation group,the difference was significant(P<0.01);after medication intervention,the protein levels of each medication group were higher than the model group,the difference was significant(P<0.05)The difference between the high-dose Juantong Decoction group was the most significant(P<0.01);the two-to-two comparisons between the drug groups showed that the PERK protein in the high-dose Juantong Decoction group was different from that of the low-dose group(P<0.05).Compared with the low-dose group and gestrinone group,the protein expression of eIF2α in the high-dose and middle-dose groups of Chinese traditional medicine was different(P<0.05).Compared with the other groups,the protein expression of LC3B in the high-dose group of Juantong Decoction had Significant difference(P<0.01),the other groups were compared in pairs,and the difference was not significant(P>0.05).(7)Results of real-time fluorescent quantitative PCR:PERK,eIF2α,and LC3B mRNA expressions in the EMs model group were significantly lower than those in the sham operation group,with significant differences(P<0.05);after medication intervention,Juantong Decoction,gestrinone and Compared with the model group,the expressions of PERK,eIF2α,and LC3B mRNA were all increased in the ERS group and significantly(P<0.05).There was a significant difference between the high and medium dose groups of Juantong Decoction(P<0.001);each drug group was compared in pairs.The LC3B and eIF2α mRNAs of the high-dose Juantong Decoction group were significantly different from those of other groups(P<0.05),and the high-dose group and the low-dose group were significantly different(P<0.01).The remaining groups were paired with each other.In contrast,there was no significant difference(P>0.05).Compared with the model group,the expression of PERK and eIF2αLC3B mRNA was up-regulated in the ERS group and there was a significant difference(P<0.05).Conclusions:1 2-DG induces an endoplasmic reticulum stress response in EMs rats,and the state of endoplasmic reticulum stress facilitates activation of the UPR and thus cellular autophagy,which clears misfolded proteins from ectopic endothelial tissue and restores homeostasis of the pelvic endotropic environment;2 Experimental evidence of a sustained inflammatory response in the pelvis of rats in the EMs model group,where high estrogen concentrations may inhibit endoplasmic reticulum stress levels,thereby blocking the PERK/eIF2αpathway,reducing the expression of autophagy-related factors in ectopic endothelium and promoting ectopic endothelial proliferation,adhesion and invasion;3 Remission drink can reduce the level of CRP and E2 in the serum of rats,inhibit the growth of pelvic ectopic lesions,reduce pelvic adhesions and improve the inflammatory response of the pelvic microenvironment;4 Juantong Decoction can up-regulate PERK,eIF2α,LC3B gene and protein expression in ectopic endometrium of EMs rats,thereby activating the endoplasmic reticulum stress response of intimal tissue,regulating PERK/eIF2α pathway to activate autophagy level,and promoting ectopic endometrial Apoptosis of endometrial cells achieves the purpose of treating EMs. |
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