| Objective:Premature ovarian failure refers to a series of perimenopausal diseases such as steroid hormone disorder,amenorrhea,and infertility caused by various factors in women before the age of 40,which seriously affects the physical and mental health of patients.In recent years,hormone replacement therapy（HRT）is the main treatment for POF and is helpful for alleviate the symptoms of the lack of estrogen,but does not fundamentally solve the result of the female reproductive.To explore a new treatment to fundamentally solve the patient’s reproductive problems become a focus and difficulty for researchers.SIRT6 is a key member of Sirtuins family,a NAD+dependent deacetylase which plays an important role in maintaining genomic stability,metabolic regulation and aging.NF-κB family are nuclear transcription factors which is widely expressed in tissues and organs.As aging occurs,the expression level of NF-κB gradually increased,so inhibiting the expression of NF-κB plays a positive role in delaying aging.A large number of studies have found that SIRT6/NF-κB signaling pathway plays an active role in various diseases such as tumor-related,inflammation-related and aging-related diseases.However,the effect of SIRT6/NF-κB signaling pathway on POF has not been systematically reported.In this study,a BALB/c mice model of POF was successfully constructed by injecting D-gal solution which is helpful for exploring the funtion and mechanism of SIRT6/NF-κB signaling pathway in POF,and aim to provide experimental basis for the treatment of POF.Methods:1.The establishment and identification of POF mouse model:42 female mice were randomly divided into control group and model group,each group of 21.Mice in the model group were injected with 200 mg·kg-1·d-1 D-gal solution daily with the back of the neck,the control group was given the same concentration of PBS buffer solution,smear vaginal exfoliated cells of mice and record estrus cycle,and record the body weight,for 42 days.Serum concentrations of estradiol（E）,follicle-stimulating hormone（FSH）and luteinizing hormone（LH）were tested by enzyme-linked immunoassay（ELISA）.Serum SOD and CAT levels were determined by the same method（ELISA）.The wet ovarian was weighed in order to calculate the ovarian coefficient.HE staining was used to observe the morphological changes of ovaries and to count the follicles at each level.The aging of the ovaries was easily find of two groups byβ-galactosidase staining.2.Exploring the SIRT6/NF-κB signaling pathway in the process of ovarian aging:（1）Quantitative real-time PCR was used to determine the expression of SIRT6 and NF-κB m RNA in ovaries of the two groups.（2）The expressions of SIRT6 and NF-κB proteins in the two groups were detected by Western-blot technique.Results:1.Compared with the control group,the body weight of mice in the model group was decreased significantly（P=0.009）;proestrus,metestrus,diestrus and estrus cycle are prolonged（P=0.012,P=0.005,P=0.007,P=0.000）,estrus was shorten;and the serum levels of E was decreased（P=0.005）,but the levels of FSH and LH were increased（P=0.005,P=0.009）,the levels of SOD and CAT were decreased（P=0.005,P=0.004）.Compared with the control group,the ovarian coefficient was decreased in the model group.By HE staining,we observed that the ovaries in the model group were significantly shrunken,and there are numerous atretic follicles.According to the result of statistic analysis find that the number of primordial,primary,secondary,and all follicles were decreased in model group（P=0.000,P=0.006,P=0.000,P=0.009）,the number of atretic follicles were increased（P=0.06）.The results ofβ-galactosidase staining showed that,galactosidase cells were significantly increased in the ovaries of the model group,and dyeing deeper compared with control group.2.Compared with the control group,the levels of SIRT6 m RNA and protein in ovary tissues of model mice were decreased（P=0.000,P=0.000）,but the levels of NF-κB m RNA and protein were increased（P=0.00,P=0.000）.Conclusions:1.POF model was successfully constructed in mice by injecting D-gal.2.SIRT6/NF-κB signaling pathway is one of the regulation mechanisms of POF. |
