Experimental Study Of CD4~+CD25~+Treg In Immunity Premature Ovarian Insufficiency Mice Therapy

Primary ovarian failure(POF),also called premature ovarian insufficiency(POI),is one of the most common reproductive disorders.POI refers to a functional decline in the female gonads characterized by the ovaries lacking functional follicles,along with increased gonadotropin levels and decreased estrogen levels in women younger than forty years old accompanied by symptoms of menopausal transition,such as menoxenia,amenorrhea,hot flashes,mood changes,bosom frowsty,and insomnia.It has been reported that the incidence of premature ovarian insufficiency is 1%before the age of forty and 0.1%before the age of thirty.In recent years,according to some study results,autoimmunity is responsible for approximately 4-30%of POI cases.At present,the treatment of POI is limited and ineffective,so it is of great significance to find a safe and effective treatment.CD4~+CD25~+regulatory T cells(Tregs)mediate immune suppression and maintain the immune homeostasis among lymphocyte subsets.They can exert immunosuppressive effects by inhibiting effector CD4~+T cells and secreting TGF-?.A reduction in Treg numbers or impairment in Treg functions can destroy self-tolerance,causing an immune overreaction and leading to the occurrence of autoimmune diseases.Interestingly,we investigated the changes of CD4~+CD25~+Tregs in POI patients and in the 3-day old mice thymectomy(D3tx)POI models.We found that the number of CD4~+CD25~+Tregs in the peripheral blood was significantly reduced in the POI patients,T cell subgroups were imbalanced,the level of TGF-?was significantly decreased,while the level of IFN-?was increased.The study showed that the numbers of CD4~+CD25~+Tregs in the peripheral blood,spleen and lymph nodes were decreased and the proportions of T lymphocyte subsets were unbalanced in D3tx POI model mice.Adoptive transfer of CD4~+CD25~+Tregs could effectively alleviate D3tx POI model mice by increasing the numbers of Tregs and regulating the proportions of T lymphocytes.CD4~+CD25~+Tregs play an important role in the development of POI.Therefore,BALB/c mice were immunized with p ZP3 polypeptide to establish an animal model of premature ovarian failure,and the therapeutic effect of CD4~+CD25~+Treg cells on premature ovarian failure mice was preliminarily explored.The research contents are as follows:1?Establishment of the model of POI.The amino acid sequence of the murine ZP3 330–342 peptide used in this study was NSSSSQFQIHGPR.The purity is 95%.The pZP3 lyophilized powder was dissolved in sterilized double distilled water and then emulsified in an equal volume of CFA(Mycobacterium tuberculosis H37RA strain).0.1ml emulsion containing 0.16 mg M.tuberculosis and 50 nmol/L p ZP3 subcutaneous injected into the hind footpad and tail root.The same immunization method was repeated with pZP3 emulsified in IFA(M.tuberculosis H37RA strain,0.16 mg/mouse)two weeks later.After reimmunization for one week,mice could be used for experimental detection.Vaginal smears of normal mice and p ZP3 model mice were observed from 8:00-9:00am every day for two weeks.The anti-zona pellucida antibodies(AZPAb)concentration and estradiol(E2),follicle-stimulating hormone(FSH),luteinizing hormone(LH)and the change of Anti Mullerian hormone(AMH)level in serum by Enzyme Linked Immunosorbent Assay(ELISA)kit detected.The ovarian appearance was observed and the ovarian sections were stained with H&E for histopathological examination by light microscopy and the follicles at different levels were counted.Immunohistochemical detected the expression of apoptosis related proteins BAX?BCL-2 and CASPASE3 in ovaries and the infiltration degree of lymphocyte.Real-time PCR and Western Blot were used to detect the expression of molecules in the TGF-?/Smads signaling pathway?PI3K/AKT/FOXO3a signaling pathway and Hippo signaling pathway in the ovary.Experimental results:(1)The p ZP3 model group had abnormal estrous cycle.After immunized with pZP3,the serum concentrations of AZPAb?FSH and LH were increased,while the serum concentrations of E2 and AMH were decreased.(2)In p ZP3model group,the ovarian size of mice was smaller and the color was white or light pink,smooth in surface,while those in the control group were ruddy and showed transparent bulges with different sizes,and the ovarian weight was significantly higher than that of the p ZP3 model group.(3)HE staining showed complete and regular ovarian morphology in the control group,in which follicles of different developmental stages were well developed,clearly visible and with a large number.In the p ZP3 model group,ovarian morphology structure was disordered,follicles were irregular and the number of follicles was significantly reduced,or even no significant follicles.Through the statistics of all levels of follicles,it was found that the primordial follicles,primary follicles and secondary follicles significantly decreased and atretic follicles increased in the pZP3 model group.The results were statistically significant between the two groups.(4)Immunohistochemical results showed that the positive expressions of CD3 and CD19 were enhanced in ovarian tissues,indicating increased lymphocyte infiltration in the ovary.The expression of BAX?CASPASE3 increased and the expression of BCL-2decreased,indicating the increase of apoptosis in ovarian tissues.(5)Real-time PCR and Western Blot results showed that the expression of TGF-?/Smads signaling pathway molecules Smad2?Smad3 and TGF-?expression were decreased,the expression of PI3K/AKT/FOXO3a signaling pathway molecules PI3K?Akt?FOXO3a were increased,and the expression of molecules Mst1/2?Lats1?Yap in Hippo signaling pathway were up-regulated.2.Mechanism of CD4~+CD25~+Treg cells in the treatment of POI.Firstly,magnetic bead sorting kit was used for sorting CD4~+CD25~+Treg from 8 weeks normal female mice spleen tissue,and the percentage of CD4~+CD25~+Treg cells was detected by Flow Cytometry.The purity of CD4~+CD25~+Treg cells was 93.2%.5x10~5CD4~+CD25~+Treg cells were adoptive transfer to POI mice.Four weeks later,ELISA kit was used to detect the serum concentration of AZPAb and the levels of LH?FSH?E2and AMH.HE was used to detect ovarian histological changes and count follicles at different development.Immunohistochemistry was used to detect the expression of BCL-2,BAX and CASPASE3 in the ovary.The tdTomato-Treg cells with red fluorescent protein were used to locate adoptive Treg cells in POI model mice.Real-time PCR was used to detect the expression of molecules in the TGF-?/Smads signaling pathway?AKT/FOXO3a signaling pathway and Hippo signaling pathway in the ovary.Experimental results:(1)The serum AZPAb concentration,FSH and LH levels were decreased,while E2 and AMH levels increased in the CD4~+CD25~+Treg cells treatment group.Compared with p ZP3 model mice,less inflammatory cells infiltrating the follicles were observed and small amounts of developed follicles presented and the number of follicles were increased.(2)The expression of apoptosis-related proteins BAX and CASPASE3 were decreased,the expression of BCL-2 was increased.(3)Immunofluorescence showed that td-Tomato-CD4~+CD25~+Treg cells transplant into the POI mice,after 24h,red fluorescence was observed in the ovarian interstitial region of POI mice.(4)Real-time PCR and immunohistochemistry results showed that the expressions of AKT and FOXO3a in ovarian tissues were down-regulated after transfer CD4~+CD25~+Treg cells,and there was no significant difference in TGF-?/Smads signaling pathway and Hippo signaling pathway.In summary,the typical characteristics of p ZP3 immune mice were similar to the POI with humans.the increase of serum autoantibodies,inflammatory cells infiltration ovarian tissue,ovarian structure disorder.The p ZP3 immune-induced POI may due to anti-ZP3 antibodies attack the zona pellucida of the oocyte result in the number of follicles decrease.It may be related to increased ovarian apoptosis events and to increased the molecules Mst1/2?Lats1 and Yap in the Hippo signaling pathway and PI3K?AKT?FOXO3a in the PI3K/AKT/FOXO3a signaling pathway,while to decreased the molecules Smad2?Smad3 and TGF-?in the TGF-?/Smads signaling pathway.Adoptive transfer CD4~+CD25~+Treg cells returned to the ovarian interstitial region in the POI mice and could effectively alleviate the symptoms,improved the serum of AZPAbs concentration and hormone level,reduced the expression of apoptosis proteins and decreased the expression of AKT and FOXO3a in the POI mice ovary.That's to provide experimental basis for clinical exploration of emerging therapeutic approaches with POI.