| Purpose:In this experiment,the rats with decreased ovarian reserve function induced by cyclophosphamide were used as the model,and the effects of traditional Chinese medicine compound,acupuncture,combination of acupuncture and traditional Chinese medicine,and western medicine were respectively used as the intervention factors,to observe the effects of autophagy related factors Atg7,LC3? and LC3? protein expression and mRNA expression of Atg7 and LC3 in ovarian granulosa cells,and to explore the mechanism of the effect of combination of acupuncture and traditional Chinese medicine therapy on the ovarian function of rats with decreased ovarian reserve function.Material and method:Sixty healthy SD female rats aged 8-9 weeks were selected and divided into 6 groups according to random table method:blank group(A group),model group(B group),western medicine group(C group),traditional Chinese medicine group(D group),acupuncture group(E group)and combination of acupuncture and traditional Chinese medicine(F group),ten in each group.All the groups except A group were modeled by intraperitoneal injection of cyclophosphamide(50mg/kg/d)for one week,and the estrous cycle was observed through the morphological changes of vaginal cells.If a certain period of time is prolonged and there is a manifestation of periodic disorder,it is proved that the modeling is successful.The intervention methods of western medicine,traditional Chinese medicine,acupuncture,combination of acupuncture and traditional Chinese medicine were applied to the successful model-making rats.The dosage of western medicine was0.78mg/100 g,and it was gavaged continuously for 3 weeks.The dose of Chinese medicine was 0.69g/kg by gavage for 3 weeks.Acupuncture was performed with 0.18×10mm acupuncture needles.Acupoints were selected as Guanyuan,Zhongji,bilateral Taichong and bilateral Taixi,once a day,and the needles were kept for 20 min for total 3 weeks.After treatment,the general behavioral changes of rats and the estrous cycle changes were observed.The morphological changes of ovarian tissue were observed by hematoxylin-eosin staining(HE).Serum AMH content of rats was determined by enzyme-linked immunosorbent assay(ELISA).Western blotting(WB)was used to detect the protein expressions of autophagy related factors Atg7,LC3? and LC3? in the ovary of rats.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the mRNA expression levels of autophagy-related factors Atg7 and LC3 in the ovary of rats.Results:1.The changes of general behavior in rats: Before the experiment,the rats in each group had normal diet,drinking water,two stools,normal activity,good mental state and bright fur.The rats in A group had no abnormal changes before and after the experiment.After modeling,the rats in each group did not want to eat and drink,their activities decreased,their mental state was poor,and their fur was dull and accompanied by hair loss.After the intervention factors were applied,diet,drinking water and defecation of rats in the treatment group gradually returned to normal,activity increased gradually,mental state improved gradually,hair color turned brighter and hair depilation decreased gradually.The overall improvement degree of the treatment group was better than that of D group and E group in F group and C group.2.The changes of estrous cycle in rats: Before the experiment,the estrous cycle of rats in each group was normal.The estrous cycle of rats in A group was in the normal range before and after the experiment,and the vaginal smear of rats in each group showed prolonged estrous cycle and disordered estrous cycle.3.The morphology of ovarian tissue in rats were observed by HE staining as follows: In A group,ovarian structure was clear,follicles at all levels developed normally with different shapes and sizes,luteal development was well developed,and follicular granulosa cell layer was abundant and uniformly arranged.In B group,ovarian atrophy was observed,the number of follicles and luteal body decreased,the number of atretic follicles increased,and the arrangement of follicular granulosa cell layer was disordered.After the intervention,the development of follicles and corpus luteum in treatment group was better than that in B group,the number of atretic follicles decreased,and the granulosa cell layer of follicles increased and arranged neatly.The overall improvement degree of the treatment group was better than that of D group and E group in F group and C group.4.Serum AMH content of rats was detected by Elisa as follows: After modeling,compared with A group,the serum AMH content of B group was decreased(P<0.01).After treatment,compared with B group,the content in the treatment group was significantly increased(P<0.01),and the content in F group was higher than that in D group and E group(P<0.05),but there was no significant difference between F group and C group(P>0.05).5.Western blotting method was used to detect the protein expression levels of autophagy related factors Atg7,LC3? and LC3? in the ovary of rats.The results were as follows: After modeling,compared with A group,the protein expression levels of Atg7 in B group were decreased(P<0.05).After treatment,compared with B group,the protein expression levels of Atg7 in treatment group were significantly increased(P<0.05),and the protein expression levels of F group were higher than those of D group and E group(P<0.05).There was no significant difference in the protein expression levels of Atg7 between F group and C group(P>0.05).After modeling,compared with A group,the protein expression levels of LC3? in B group were decreased(P<0.05).After treatment,compared with B group,the protein expression levels of LC3? in treatment group were significantly increased(P<0.05),and the protein expression levels of F group were higher than those of D group and E group(P<0.05).There was no significant difference in the protein expression levels of LC3? between F group and C group(P>0.05).After modeling,compared with A group,the protein expression levels of LC3? in B group were increased(P<0.05).After treatment,compared with B group,the protein expression levels of LC3? in treatment group were significantly decreased(P<0.05),and the protein expression levels of F group were higher than those of D group and E group(P<0.05).There was no significant difference in the protein expression levels of LC3? between F group and C group(P>0.05).6.The mRNA expression levels of autophagy-related factors Atg7 and LC3 in the ovary of rats were detected by qRT-PCR.The results showed that: After modeling,compared with A group,the mRNA expressions of Atg7 in B group were decreased(P<0.05).After treatment,compared with B group,the mRNA expression levels of Atg7 in the treatment group were significantly increased(P<0.05),the mRNA expression levels in F group were higher than those in D group and E group(P<0.05),and there was no significant difference in the mRNA expression levels between F group and C group(P>0.05).After modeling,compared with A group,the mRNA expressions of LC3 in B group were decreased(P<0.05).After treatment,compared with B group,the mRNA expression levels of LC3 in the treatment group were significantly increased(P<0.05),the mRNA expression levels in F group were higher than those in D group and E group(P<0.05),and there was no significant difference in the mRNA expression levels between F group and C group(P>0.05).Conclusion:1.Combination of acupuncture and traditional Chinese medicine,traditional Chinese medicine compound and acupuncture can effectively improve the pathological state and ovarian tissue morphology of rats with decreased ovarian reserve.2.Combination of acupuncture and traditional Chinese medicine can effectively improve the pathological state and ovarian tissue morphology of rats with decreased ovarian reserve,and its curative effect is better than that of traditional Chinese medicine or acupuncture therapy.3.Combination of acupuncture and traditional Chinese medicine can increase the expression of Atg7 and LC3? and decrease the expression of LC3? in rats with decreased ovarian reserve.The mechanism may be that it interfered with the autophagy process of ovarian granular cells. |
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