Mechanisms Of GADD45b In The Repair Of Chemotherapy-induced Premature Ovarian Failure In Mice By MB-MSCs

Chemotherapy is one of the important means of comprehensive treatment of malignant tumors and immune diseases,but the selectivity of chemotherapeutic drugs to target cells is poor,while killing pathological cells,normal tissues and cells also have varying degrees of damage.Especially with the younger lymphoma and breast cancer patients and the higher survival rate,the iatrogenic damage caused by chemotherapeutic drugs can not be ignored.Epirubicin,as a representative of anthracycline antibiotics,is the first choice of first-line chemotherapeutic drugs for female breast cancer,ovarian cancer,leukemia and childhood lymphoma.Its antineoplastic spectrum is wide,its cardiotoxicity is low,and its curative effect is affirmative.However,it is reported that 60%of adult women will suffer from ovarian dysfunction after epirubicin chemotherapy,including menstrual disorders,amenorrhea,infertility,and even premature ovarian failure?POF?^[1].The mechanism and treatment of POF is a hot topic for researchers and clinicians nowadays.Repair of chemotherapy-induced premature ovarian failure?CIPOF?by endometrial mesenchymal stem cells isolated from menstrual blood?MB-MSCs?is a new research direction.However,the specific mechanism of action is not yet clear.OBJECTIVE:In our previously vitro experiments,we used Affymetrix Gene Chip to analyze the whole genome transcription of human ovarian granulosa cells?GCs?before and after treatment with epirubicin.We found that GADD45b was one of the differentially expressed genes.The down-regulation of GADD45b and the up-regulation of CYCLINB1 and CDC2 may be the core mechanism of MB-MSCs to repair epirubicin-induced GCs damage^[2].This study will further explore whether MB-MSCs can repair CIPOF by altering the molecular mechanism of GADD45b gene network in mice.METHODS:CIPOF mice model was established by intraperitoneal injection of epirubicin.MB-MSCs were injected into tail vein as an intervention method,and the estrous cycle of mice was observed continuously every day.After 28 days of MB-MSCs transplantation,the levels of serum E2,FSH and AMH were determined by ELISA,follicular development was observed by HE staining,and the apoptosis of ovarian tissue in situ was detected by TUNEL.The expression levels of GADD45b,CDC2 and CYCLINB1 in ovarian tissues of mice were detected by Western Blot and RT-PCR.RESULTS:CIPOF mouse model was successfully constructed.28 days after transplantation of MB-MSCs,ELISA results showed that the serum FSH level in POF group?model group?was higher than that in Control group?normal control group??327.817±12.627 vs 163.152±4.961,P<0.05?,AMH and E2 levels were lower?1138.575±50.246 vs 2760.227±128.140,P<0.05;43.772±3.407 vs 117.784±4.821,P<0.05?,the differences were statistically significant;Compared with POF group and Control group,FSH?259.614±3.588?,AMH?1920.016±184.215?and E2?83.077±10.882?in POF+M group?stem cell therapy group?had significant difference?P<0.05?.HE staining showed that the number of primordial follicles?17.333±4.190??primary follicles?8.333±2.055??secondary follicles?6.667±2.055??and antral follicles?2.000±0.816?in POF group was the least among the three groups,while the number of atresic follicles?146.000±5.888?was the most;The number of primordial follicles?42.667±3.300??primary follicles?18.667±1.247??secondary follicles?19.333±1.247??and antral follicles?8.333±1.886?in POF+M group was higher than that in POF group?P<0.05?,but still less than that in Control group?70.000±5.099,30.667±3.091,44.333±6.128,21.667±9.177??P<0.05?,the number of atresia follicles?86.667±4.784?was lower than that in POF group?P<0.05?,but still more than that in Control group?28.333±9.177??P<0.05?,the differences were statistically significant.At thes ame time,microscopically,the ovarian interstitial vessels in POF group were less than those in Control group,and the cortex was thinner.TUNEL results showed that the percentage of TUNEL-positive cells?21.129±2.398?%in ovarian tissue of POF+M group was lower than that in POF group?40.676±3.782?%?P<0.05?,but was still higher than that in Control group?4.346±0.888?%?P<0.05?,the difference was statistically significant;the percentage of TUNEL-positive cells in POF group was higher than that in Control group?P<0.05?,the difference was statistically significant.Western Blot and RT-PCR results showed that the expression of GADD45b in POF group increased?P<0.05?,and the expression of CYCLINB1 and CDC2 decreased?P<0.05?compared with that in Control group,the differences were statistically significant;Compared with POF group,the expression of GADD45b in POF+M group decreased?P<0.05?,and CYCLINB1 and CDC2 increased?P<0.05?,the differences were statistically significant.CONCLUSION:We successfully induced CIPOF mice models with epirubicin.MB-MSCs transplantation can reduce FSH and increase AMH and E2 in serum of mice in POF group;increase the number of primordial follicles?primary follicles?secondary follicles and antral follicles,decrease the number of atresia follicles;decrease the apoptosis of ovary in situ;decrease the expression of GADD45b,and increase the expression of CYCLINB1 and CDC2.MB-MSCs can protect the ovarian structure and function of mice from epirubicin-induced damage.The molecular mechanism may be that MB-MSCs can repair CIPOF by down-regulating GADD45b expression,up-regulating CYCLINB1 and CDC2 expression,regulating cell cycle,inhibiting cell apoptosis.