| ObjectiveStarting from autophagy,the mechanism of electroacupuncture regulating the endometrial autophagy of intrauterine adhesion(IUA)rat was elucidated,in order to provide experimental evidence for the study of intrauterine adhesion by electroacupuncture.MethodsExperiment 1 Effect of electroacupuncture on the repair of endometrial damage in rats with IUA:forty-five SPF 6-8 week-old female SD rats were randomly divided into three groups according to the random number table method,with 15 rats in each of the three group:control group,IUA group and electroacupuncture group(EA group).The IUA rat model was established by mechanical injury plus lipopolysaccharide(LPS)infection.On the next day of molding,"Guanyuan"(CV4)"Sanyinjiao"(SP6)and "Zusanli"(ST36)were administered.Bilateral SP6 and ST36 were connected to an electrical stimulator.The intensity of stimulation should be the slight trembling of the rat’s limbs.The stimulation was conducted once daily for 15 min for three consecutive estrus cycles.The control and model rats were fixed without acupuncture intervention.Five female rats were mated with male rats in a ratio of 2:1 during estrus.The morning of the occurrence of vaginal plugs was considered as the day 1 of gestation.On the 8th day of gestation,the number of embryos implanted in the rat was calculated.Ten remaining female rats were sacrificed after isoflurane overdose anesthesia in the third estrous period after modeling,and uterine tissues were collected.The alteration of endometrial morphology was observed by HE staining,while the region of uterine fibrosis was determined by Masson staining.The expression of transforming growth factor-β1(TGF-β1)and α-smooth muscle actin(α-SMA)in rat endometrial tissues was detected by immunohistochemistry method.The protein expression of homeobox A10(HoxA10)and leukemia inhibitory factor(LIF)in rat endometrial tissues was detected by Western Blot method.The gene expression of HoxA10 and LIF mRNA in rat endometrial tissues was detected by qPCR method.Experiment 2 Electroacupuncture activated endometrial autophagy in rats with IUA:the experimental animals and method are the same as experiment 1.The ultrastructure of autophagosomes in endometrial epithelial cells was observed by transmission electron microscopy(TEM).The protein and gene expression of microtubule-associated protein 1 light chain 3(LC3),Beclinl and p62 in rat endometrial tissues was detected by immunohistochemistry,Western Blot and qPCR method.Experiment 3 Electroacupuncture inhibited endometrial inflammation in rats with IUA:the experimental animals and method are the same as experiment 1.The protein expression of inflammation-related factors interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)in rat endometrial tissues was detected by ELISA method.The protein expression of p65,inhibitor of KappaB Kinase alpha(IKKα)and inhibitor kappa B alpha(IκBα)in rat endometrial tissues was detected by Western Blot method.The gene expression of IL-1β,IL-6 and TNF-α mRNA in rat endometrial tissues was detected by qPCR method.Experiment 4 Mechanism of electroacupuncture regulating autophagy inhibiting endometrial inflammation in rats with IUA:forty SPF 6-8 week-old female SD rats were randomly divided into four groups,with 10 rats in each of the four group:IUA group,electroacupuncture group,electroacupuncture+inhibitor and agonist group.The modeling method and electroacupuncture intervention were the same as experiment 1.The electroacupuncture+inhibitor group was injected intraperitoneally with autophagy inhibitor 3methyladenine(3-MA),1.5 mg/kg/d on the basis of electroacupuncture therapy,for 7 days.The agonist group was injected intraperitoneally with autophagy agonist rapamycin(RAPA),2 mg/kg/d,for 7 days.All rats were sacrificed after isoflurane overdose anesthesia in the third estrous period after modeling,and uterine tissues were collected.The alteration of endometrial morphology was observed by HE staining,while the region of uterine fibrosis was determined by Masson staining.The ultrastructure of autophagosomes in endometrial epithelial cells was observed by TEM.The protein expression of LC3,Beclinl,p62 as well as TGF-β1,α-SMA in rat endometrial tissues was detected by immunohistochemistry.The protein expression of LC3,Beclinl,p62 as well as p65,IKKα,IκBα in rat endometrial tissues was detected by Western Blot.The gene expression of LC3,Beclinl,p62 as well as IL-1β,IL-6,TNF-α mRNA in rat endometrial tissues was detected by qPCR.Results1.Effect of electroacupuncture on the repair of endometrial damage in rats with IUA(1)The change of endometrial morphology was observed by HE stainingCompared with the control group,the endometrial glands in the model group were decreased(P<0.01),and the endometrium was thinner(P<0.01).After electroacupuncture treatment,the number of endometrial glands was significantly increased(P<0.01),and the endometrium was thicker(P<0.05).(2)The area of uterine fibrosis was calculated by Masson stainingThe endometrial fibrosis of model group was more serious than that of control group(P<0.01).Electroacupuncture treatment alleviated the degree of endometrial fibrosis(P<0.05).(3)The protein expression of TGF-β1 and α-SMA in endometrial tissue of rats was detected by immunohistochemistry methodThe expression intensity of TGF-β1 and α-SMA in the model group was increased compared with the control group(P<0.01,P<0.01),yet the expression intensity of TGF-β1 andα-SMA was decreased after electroacupuncture treatment(P<0.05,P<0.05).(4)The protein expression of HoxA10 and LIF in endometrial tissue of rats was detected by Western Blot methodThe protein expression of HoxA10 and LIF in the model group was significantly reduced compared with the control group(P<0.01,P<0.01),yet the expression of HoxA10 and LIF protein was increased after electroacupuncture treatment(P<0.01,P<0.01).(5)The gene expression of HoxA10 and LIF mRNA in endometrial tissue of rats was detected by qPCR methodThe mRNA expression of HoxA10 and LIF in the model group was down-regulated compared with those in the control group(P<0.01,P<0.01),yet the mRNA expression of HoxA10 and LIF was up-regulated after electroacupuncture treatment(P<0.05,P<0.01).(6)Embryos implantation of rats in each groupThe number of implanted embryos in the control group was the highes,there were no embryos implantation in the damaged uterus in the model group(P<0.01).In the electroacupuncture group,the number of implanted embryos on the damaged side was increased(P<0.01).2.Electroacupuncture activated endometrial autophagy in rats with IUA(1)The ultrastructure of autophagosomes in endometrial epithelial cells was observed by TEM.There were abundant autophagosomes in the control group,the number of autophagosomes in the model group was reduced compared with the control group.After electroacupuncture treatment,there were more autophagosomes.(2)The expression of LC3,Beclinl and p62 in rat endometrial tissues was detected by immunohistochemistry methodThe expression intensity of LC3 and Beclinl was weaken in the model group compared with the control group(P<0.01,P<0.01),the expression intensity of p62 was enhanced(P<0.01).After electroacupuncture treatment,the expression intensity of LC3 and Beclinl was enhanced(P<0.01,P<0.05),and the expression intensity of p62 was weaken(P<0.05).(3)The protein expression of LC3,Beclinl and p62 in rat endometrial tissues was detected by Western Blot methodThe protein expression of LC3 and Beclinl in the model group was significantly decreased compared with the control group(P<0.01,P<0.01),the p62 protein expression level was significantly increased(P<0.01).After electroacupuncture treatment,the protein expression of LC3 and Beclinl was significantly increased(P<0.01,P<0.01),the p62 protein expression level was significantly decreased(P<0.01).(4)The gene expression of LC3,Beclinl and p62 mRNA in rat endometrial tissues was detected by qPCR methodThe gene expression of LC3 and Beclinl mRNA in the model group was down-regulated compared with the control group(P<0.01,P<0.01),the gene expression of p62 mRNA was upregulated(P<0.01).After electroacupuncture treatment,the gene expression of LC3 and Beclinl mRNA was up-regulated(P<0.01,P<0.01),the gene expression of p62 mRNA was down-regulated(P<0.01).3.Electroacupuncture inhibited endometrial inflammation in rats with IUA(1)The protein expression of inflammation-related factors IL-1β,IL-6 and TNF-α in rat endometrial tissues was detected by ELISA methodThe protein expression of IL-1β,IL-6 and TNF-α in the model group was increased compared with the control group(P<0.01,P<0.01,P<0.01),yet the protein expression of IL-1β,IL-6 and TNF-α was decreased after electroacupuncture treatment(P<0.01,P<0.01,P<0.01).(2)The protein expression of NF-κB signal pathway related protein p65,IKKα and IκBαin rat endometrial tissues was detected by Western Blot methodThe phosphorylation level of p65,IKKa and IκBα in model group was up-regulated compared with the control group(P<0.01,P<0.01,P<0.01),yet the phosphorylation level of p65,IKKα and IκBα was significantly down-regulated after electroacupuncture treatment(P<0.01,P<0.01,P<0.01).(3)The gene expression of IL-1β,IL-6 and TNF-α mRNA in rat endometrial tissues was detected by qPCR methodThe gene expression of IL-1β,IL-6 and TNF-α mRNA in the model group was significantly up-regulated compared with the control group(P<0.01,P<0.05,P<0.01).The gene expression of IL-1β,IL-6 and TNF-α mRNA was down-regulated after electroacupuncture treatment(P<0.01,P<0.05,P<0.01).4.Mechanism of electroacupuncture regulating autophagy inhibiting endometrial inflammation in rats with IUA(1)Endometrial autophagy of rats in each group① The ultrastructure of autophagosomes in endometrial epithelial cells was observed by TEMIn the model group,autophagosomes in endometrial epithelial cells were less.There were more autophagosomes in the electroacupuncture and agonist groups,and the nuclear membrane was relatively intact.The autophagosomes in the electroacupuncture+inhibitor group were less.② The expression of LC3,Beclinl and p62 in rat endometrial tissues was detected by immunohistochemistry methodThe expression intensity of LC3 in the electroacupuncture and agonist groups was enhanced than that in model group(P<0.01,P<0.05),yet there was no significant difference between the electroacupuncture+inhibitor group and model group(P>0.05).The expression intensity of LC3 in electroacupuncture+inhibitor group was weaken compared with that in electroacupuncture group(P<0.01),but there was no statistical significance between agonist group and electroacupuncture group(P>0.05).The expression intensity of Beclinl in the electroacupuncture and agonist groups was enhanced than that in model group(P<0.01,P<0.01),yet there was no significant difference between the electroacupuncture+inhibitor group and model group(P>0.05).The expression intensity of Beclinl in electroacupuncture+inhibitor group was weaken compared with that in electroacupuncture group(P<0.05),but there was no statistical significance between agonist group and electroacupuncture group(P>0.05).The expression intensity of p62 in the electroacupuncture and agonist groups was weaken compared with the model group(P<0.01,P<0.05),yet there was no significant difference between the electroacupuncture+inhibitor group and model group(P>0.05).The expression intensity of p62 in electroacupuncture+inhibitor group was enhanced compared with the electroacupuncture group(P<0.01),there was no statistical significance between agonist group and electroacupuncture group(P>0.05).③The protein expression of LC3,Beclinl,and p62 in rat endometrial tissues was detected by Western Blot methodThe protein expression of LC3 was increased in the electroacupuncture and agonist group(P<0.01,P<0.01),yet there was no statistical difference between the electroacupuncture+inhibitor group and the model group(P>0.05).The protein expression of LC3 in the electroacupuncture+inhibitor group was decreased compared with the electroacupuncture group(P<0.01),yet there was no statistical difference between the agonist group and the electroacupuncture group(P>0.05).The protein expression of Beclinl was increased in the electroacupuncture and agonist group compared with the model group(P<0.01,P<0.01),yet there was no statistical difference between the electroacupuncture+inhibitor group and the model group(P>0.05).The protein expression of Beclinl in the electroacupuncture+inhibitor group was decreased compared with the electroacupuncture group(P<0.05),yet there was no statistical difference between the agonist group and the electroacupuncture group(P>0.05).The protein expression of p62 was decreased in the electroacupuncture and agonist group compared with the model group(P<0.05,P<0.01),yet there was no statistical difference between the electroacupuncture+inhibitor group and the model group(P>0.05).The protein expression of p62 in the electroacupuncture+inhibitor group was increased compared with the electroacupuncture group(P<0.01),yet there was no statistical difference between the agonist group and the electroacupuncture group(P>0.05).④ The gene expression of LC3,Beclinl and p62 mRNA in rat endometrial tissues was detected by qPCR methodThe gene expression of LC3 and Beclinl mRNA was significantly up-regulated in the electroacupuncture and agonist group compared with the model group(P<0.01,P<0.01),the gene expression of p62 mRNA was down-regulated(P<0.01),yet there was no statistical difference between the electroacupuncture+inhibitor group and the model group(P>0.05).The gene expression of LC3 and Beclinl mRNA in the electroacupuncture+inhibitor group was significantly down-regulated compared with the electroacupuncture group(P<0.01,P<0.01),the gene expression of p62 mRNA was up-regulated(P<0.01),yet there was no statistical difference between the agonist group and the electroacupuncture group(P>0.05).(2)Endometrial inflammation of rats in each group①The protein expression of NF-κB signal pathway related protein p65,IKKα and IκBα in rat endometrial tissues was detected by Western Blot methodThe phosphorylation expression of p65 protein was significantly decreased in the electroacupuncture and agonist group compared with the model group(P<0.01,P<0.01),yet there was no statistical difference between the electroacupuncture+inhibitor group and the model group(P>0.05).The phosphorylation expression of p65 protein in the electroacupuncture+inhibitor group was increased compared with the electroacupuncture group(P<0.05),yet there was no statistical difference between the agonist group and the electroacupuncture group(P>0.05).The phosphorylation expression of IKKa and IκBα protein was decreased in the electroacupuncture and agonist group compared with the model group(P<0.01,P<0.01),yet there was no statistical difference between the electroacupuncture+inhibitor group and the model group(P>0.05).The phosphorylation expression of IKKα and IκBα protein in the electroacupuncture+inhibitor group was significantly increased compared with the electroacupuncture group(P<0.01,P<0.01),yet there was no statistical difference between the agonist group and the electroacupuncture group(P>0.05).② The gene expression of IL-1β,IL-6 and TNF-α mRNA in rat endometrial tissues was detected by qPCR methodThe gene expression of IL-1β,IL-6 and TNF-α mRNA was down-regulated in the electroacupuncture and agonist group compared with the model group(P<0.01,P<0.01,P<0.01),yet there was no statistical difference between the electroacupuncture+inhibitor group and the model group(P>0.05).The gene expression of IL-1β,IL-6 and TNF-α mRNA in the electroacupuncture+inhibitor group was up-regulated compared with the electroacupuncture group(P<0.01,P<0.01,P<0.01),yet there was no statistical difference between the agonist group and the electroacupuncture group(P>0.05).(3)Endometrial repair effect in each group① The change of endometrial morphology was observed by HE stainingThe number of endometrial glands was significantly increased compared with the model group(P<0.01,P<0.01),and the endometrium became thicker(P<0.01,P<0.01),yet there was no statistical difference between electroacupuncture+inhibitor group and model group(P>0.05).The number of endometrial glands in the electroacupuncture+inhibitor group was significantly reduced compared with the electroacupuncture group(P<0.01),and the endometrium became thinner(P<0.01),yet there was no statistical difference between the agonist group and the electroacupuncture group(P>0.05).② The area of uterine fibrosis was calculated by Masson stainingThe electroacupuncture group and agonist group relieved endometrial fibrosis more than the model group(P<0.01,P<0.01),yet there was no statistical difference between the electroacupuncture+inhibitor group and the model group(P>0.05).The fibrosis was more severe in the electroacupuncture+inhibitor group than in the electroacupuncture group(P<0.01),yet there was no significant difference between the agonist group and the electroacupuncture group(P>0.05).③The expression of TGF-β1 and α-SMA in rat endometrial tissues was detected by immunohistochemistry methodThe expression intensity of TGF-β1 and α-SMA in the electroacupuncture and agonist groups was weaken compared with the model group(P<0.01,P<0.01),yet there was no significant difference between the electroacupuncture+inhibitor group and model group(P>0.05).The expression intensity of TGF-β1 and α-SMA in electroacupuncture+inhibitor group was enhanced compared with the electroacupuncture group(P<0.01),but there was no statistical significance between agonist group and electroacupuncture group(P>0.05).Conclusions1.Electroacupuncture can promote the recovery of uterine cavity morphology,increase the number of glands and the thickness of the endometrium,reduce the degree of uterine fibrosis in rats with IUA,improve the receptivity of endometrium,and facilitate embryo implantation.2.Electroacupuncture can activate autophagy in rats with IUA.3.Electroacupuncture can inhibit the inflammatory response in rats with IUA.4.Electroacupuncture may reduce NF-κB signal pathway mediated inflammation by activating autophagy,and promote the repair of endometrial damage in rats with IUA. |
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