| Background and aimsPremature Ovarian Insufficiency(Premature Ovarian Insufficiency,POI,also known as premature ovarian failure)refers to the female before 40 years occours primary or secondary amenorrhea for at least 4 months,accompanied by decreased serum estrogen levels and gonadotropin FSH level rised.At present,the etiologies of POI are diverse,including gene mutation,autoimmune disease,infectious factors,metabolic factors,iatrogenic factors,environmental pollution,psychological factors and so on.As estrogen levels decrease,elevated levels of gonadotropins often lead to a series of clinical complications,including:infertility,cardiovascular diseases,osteoporosis,urinary nervous system disorders and psychiatric symptoms,seriously affecting the normal life of women.Cisplatin(CDDP),a commonly used oncology chemotherapy drugs,can inhibit DNA replication and belongs to cytotoxic drugs.It mainly damages granulocytes and interstitial cells,leading to ovarian dysfunction and even POI.It has been reported in a small number of cases that cisplatin-induced ovarian dysfunction is associated with endoplasmic reticulum stress but the mechanism is not clear.Endoplasmic reticulum(ER)is an important organelle in eukaryotic cells involved in protein folding and processing,lipid synthesis and secretion.Endoplasmic reticulum stress(ERS)is activated when intracellular proteins folding error,infected,altered in pH,hypoxia or imbalance of Ca2+ Endoplasmic reticulum stress activates an unfolded protein response(UPR)that recognizes and cleans these misfolded proteins,stabilizes the endoplasmic reticulum,and protects cells from damage.When excessive or endoplasmic reticulum stress lasts for long time,it can lead to apoptosis.The main functions of UPR are as follows:(1)to induce the expression of endoplasmic reticulum stress protein,such as HSPA5.(2)Inhibition of protein translation.When HSPA5 binds to the unfolded protein in the endoplasmic reticulum,it binds to PERK,activating the PERK signaling pathway and promoting phosphorylation of eukaryotic translation initiation factor 2a subunit(e1F2α)Reduced protein synthesis.(3)Excessive endoplasmic reticulum stress induces CHOP expression leading to apoptosis.4-phenylbutyrate(4-PBA)can downregulate the ERS-related protein,and thus can be used as an inhibitor of endoplasmic reticulum stress to inhibit endoplasmic reticulum stress and explore the relationship between endoplasmic reticulum stress and ovarian damage.Autophagy is an intracellular degradation system that plays an important role in the regulation of intracellular homeostasis by degrading damaged or aged proteins or organelles through the lysosomal pathway when the cells are exposed to metabolic stress.Too strong cell autophagy can damage cells,triggering apoptosis.3-methyladenine(3-MA),an autophagy inhibitor that decreases autophagy,was used in our experiments to explore the mechanism of autophagy and cisplatin-induced ovarian damage.In recent years,the relationship between endoplasmic reticulum stress-induced signaling pathway and autophagic apoptosis has become a research hotspot,but its specific interaction needs further explored and verified.This issue through the establishment of cisplatin-induced mouse POI model and cisplatin in vitro stimulation of ovarian granule cells experiment.Clarified the role of autophagy and apoptosis in cisplatin-induced follicular development and ovarian dysfunction by histology and cell molecular biology methods from the molecular,cellular and overall level.The use of endoplasmic reticulum stress inhibitor 4-PBA alleviated cisplatin-induced POI and protect the ovarian damage caused by chemotherapy drugs such as cisplatin,providing a new experimental basis and potential therapeutic targets.Methods1、C57BL/6J mice were treated with cisplatin to induce mouse POI model.2、Detect ovarian tissue of C57BL/6J mice treated with cisplatin by iTRAQ proteomics technology and screen the protein with significant changes.HSPA5 and HSP90AB1 genes were screened out by bioinformatics analysis.3、The mRNA expression of HSPA5 and HSP90AB1 in ovarian tissue induced by cisplatin was verified by real-time quantitative PCR.The protein level was analyzed by western blot.4、The relationship between endoplasmic reticulum stress,autophagy and apoptosis in KGN granulosa cells and COV434 granulosa cells was analyzed by flow cytometry after cisplatin,4-PBA and 3-MA treatment.5、The effect of 4-PBA and 3-MA on cisplatin-induced autophagy of KGN granulosa cells was detected by DALgreen.6、C57BL/6J mice were treated with 4-PBA,3-MA and cisplatin for 1 day,3 days and 7 days.The morphological changes of ovarian tissue were analyzed by HE staining and immunofluorescence.Statistics of primary follicles,secondary follicles,antral follicles and atresia follicles number.7、The effect of cisplatin on apoptosis of mouse follicle cell was detected by TUNEL.8、The serum of mice treated with various treatment protocols for 7 days were analyzed by enzyme-linked immunosorbent assay(ELISA)and biochemically analyzed to detect the changes of E2 and FSH hormones in mice.Results1.Ovarian HSPA5 and HSP90AB1 protein levels were significantly reduced in the CDDP-induced POI model.In order to investigate the mechanism of cisplatin-induced POI,iTRAQ proteomics was used to analyze the protein expression of ovarian tissues in cisplatin-treated mice.A total of 5668 proteins were screened,compared with the control group,up-regulated more than 1.5-fold protein 214 species,down 1.5 times more than 180 kinds of proteins.Further analysis of these significantly altered proteins by bioinformatics revealed that the endoplasmic reticulum stress-related proteins HSPA5 and HSP90AB1 in cisplatin treated group were down-regulated 2.5-fold and 2.3-fold,respectively.To further validate the results of iTRAQ,we detected the changes of ovarian mRNA levels in cisplatin-treated mice by real-time quantitative PCR.The results also showed that the experimental group HSPA5 and HSP90AB1 mRNA levels were significantly lower than the control group,p values were p=0.003 and p=0.047.Interestingly,the ovaries after 7 days of cisplatin treatment were significantly lower in mRNA and protein levels from HSP90AB1 and HSPA5.Western blotting further confirmed the dramatic reduction in their protein levels in the CDDP group.These results indicated that ERS was strongly associated with CDDP-induced POI。2、Reducing endoplasmic reticulum stress-related genes by siRNA reduces autophagy and apoptosis levelsFirst,the immunofluorescence technique was used to detect the localization of anti-HSPA5 antibody and anti-HSP90AB1 antibody on mouse ovarian paraffin tissue sections and found to be mainly expressed in the cytoplasm of granulosa cells and the intensity of expression in apoptotic granulosa cells was normal Granulosa cells strong.It can be speculated that the occurrence of endoplasmic reticulum stress mainly in ovarian granulosa cells,so in subsequent cell experiments,we use granule cell as vitro cell experiments.In order to explore the reasonable dosages and treatment experiments of CDDP in the cell experiment,we designed the CDDP concentration gradient and the treatment time gradient.KGN granule cells were treated with 0,2,5,10,25 and 50 μM CDDP for 24 hours.Cells were harvested and the protein expression of HSP90AB1 and HSPA5 was verified by western blot.We found that the protein levels of HSP90AB1 and HSPA5 were significantly decreased when treated with cisplatin at a concentration of 50μM.Also in KGN granulosa cells,treated with cisplatin at a concentration of 50 μM for 0,4,8,16 and 24 hours,cells were collected and the protein expression of HSP90AB1 and HSPA5 was verified by western blot.The protein levels of HSP90AB1 and HSPA5 were found to rise at 4 hours with cisplatin treatment and significantly decrease after 24 hours.To investigate the effect of HSP90AB1 and HSPA5 on the autophagy and apoptosis of granulosa cells stimulated by cisplatin,we first transfected KGN granulosa cells and COV434 granulosa cells with siRNAs of Hsp90abl and Hspa5,Control group,48 hours after transfection,treated with 50μM of cisplatin 0,8,16,24 hours,and then by western blot to verify the expression of endoplasmic reticulum stress,autophagy and apoptosis in granulosa cells.Compared with the control group,the expression of HSP90AB1 and HSPA5 at each time point in the experimental group were significantly decreased,which proved that the siRNA transfection protein knockdown was effective.P6 and ATG12 as markers to detect the level of autophagy,compared with the control group,mild P62 protein and ATG12 expression decreased,indicating that knocked down endoplasmic reticulum stress can inhibit autophagy protein expression levels.As a marker of apoptosis,PARP is cleaved-PARP cleaved by apoptotic protein caspase and increased with the increase of apoptosis.The cleaved-PARP protein level in the experimental group was significantly lower than that in the control group.It has been shown that the inhibition of ER stress expression can reduce the expression of apoptotic proteins.3.4-PBA can alleviate cisplatin-induced endoplasmic reticulum stress in granulosa cells and reduce the level of autophagy and apoptosis1)The effect of 4-PBA as an endoplasmic reticulum stress inhibitor on endoplasmic reticulum stress is theoretically similar to that of Hsp90ab1 and Hspa5 siRNA.In KGN granulosa cells and COV434 granulosa cells,treated with cisplatin at a concentration of 5 mM and treated with 4-PBA for 0,4,8,16 and 24 hours,the cells were collected and verified for expression of HSP90AB1 and HSPA5 by western blot Compared with the cisplatin group alone,the level of protein expression in the treated group was still lower than that of the cisplatin group alone.The change of protein expression level was also observed after 4 hours of treatment and decreased after 24 hours.The autophagy markers P62 and ATG12 showed an increase from 8 to 16 hours after cisplatin treatment,while the expression of cleaved-PARP,an apoptosis-related protein,increased from 16 to 24 hours after cisplatin treatment.From the above experimental results,it can be speculated that cisplatin can activate endoplasmic reticulum stress in granulosa cells,enhanced endoplasmic reticulum stress can stimulate autophagy,finally induce apoptosis,enhanced autophagy and apoptosis can reduce the endoplasmic reticulum Net stress level.To further verify the effect of 4-PBA on cisplatin-induced apoptosis of KGN granulosa cells,we treated the cells with the same drug concentration for 24 hours and detected the apoptosis by flow cytometry.We found that cisplatin combined with 4-PBA Treatment,compared with cisplatin treatment alone,the rate of apoptosis decreased,4-PBA can be shown to mitigate cisplatin-induced granulosa cell apoptosis.4.Suppressing autophagy with 3-MA neither reduced CDDP-induced cell apoptosis nor alleviated ERS.In immunofluorescence assay,anti-LC3 antibody was used to detect the localization of LC3 protein in the cytoplasm of mouse granulosa cells,which was similar to the expression of endoplasmic reticulum stress.The localization of LC3 protein in the apoptotic granulosa cells LC3 expression intensity than normal granulosa cells.It can be speculated that the occurrence of autophagy is also mainly in ovarian granulosa cells.The first to dye granulosa cells with a DALGreen fluorescent dye,a dye that emits green fluorescence by binding to autophagosomes,the stronger the fluorescence intensity,the higher the level of autophagy.3-MA as an inhibitor of autophagy.The experiment was divided into four groups,namely the control group,that is,only DALGreen fluorescent dye was added as the control group.On the basis of DALGreen fluorescent dye,experimental groups were treated with cisplatin,cisplatin+4-PBA,cisplatin+3-MA(5 mM).The experimental results showed that both 4-PBA and 3-MA can reduce the level of autophagy induced by cisplatin,in contrast with the strong fluorescence intensity observed by flow cytometry or confocal microscopy,but 4-PBA reduced autophagy The effect is stronger than 3-MA.Statistics positive cells observed under confocal microscopy,compared with the cisplatin group,the control group and plus 4-PBA drug-treated group of positive cells was significantly lower,and with a statistically significant difference(p=0.024 And p=0.011).5.3-MA reduced cisplatin-induced granulosa cell autophagy,but had no significant effect on endoplasmic reticulum stress and apoptosisIn KGN granulosa cells and COV434 granulosa cells,the cells were treated with 5 mM 3-MA at the above cisplatin concentrations for 0,4,8,16,and 24 hours.The collected cells were verified by western blot to find that the level of autophagy-related proteins P62 and ATG12 was lower in the 3-MA group than the cisplatin group alone,especially ATG12.However,the expression of HSP90AB1、HSPA5 proteins and apoptosis-related protein cleaved-PARP did not show significant differences between the two groups.To further verify the effect of 3-MA on cisplatin-induced apoptosis of KGN granulosa cells,we treated with the same drug concentration for 24 hours and detected the apoptosis by flow cytometry.We found that cisplatin combined with 3-MA There was no significant difference in apoptotic rate between the treatment group and the cisplatin alone group,demonstrating that 3-MA did not significantly relieve cisplatin-induced granulosa cell apoptosis,and then deduced that autophagy could inhibit the endoplasmic reticulum stress and apoptosis Death has no significant effect.5.Effects of 4-PBA and 3-MA on CDDP-induced POI in vivoWe then compared the in vivo protective effects of 4-PBA and 3-MA against CDDP-induced ovarian damage.Female mice were treated with CDDP combined with 4-PBA or 3-MA for 1,3,or 7 days.A histological examination showed that the morphologies of follicles at different stages of development were similar across all the groups on day 1.However,on day 3,the CDDP-treated group had fewer healthy follicles(although not significantly)and significantly more atretic follicles than the saline-treated group(P<0.05),which was predominantly attributable to the loss of antral follicles.Both 4-PBA and 3-MA slightly reduced CDDP-induced follicular atresia.By day 7,the longer exposure to CDDP seriously damaged the healthy follicles and produced excessive atretic follicles(both P<0.01),because as well as antral follicles,more secondary follicle were damaged,whereas the primary follicles appeared not susceptible.Therefore,CDDP preferentially targeted and damaged mature and developing follicles rather than undeveloped follicles,supporting a previous report that chemotherapeutic agents over activate the primordial follicles and damage the growing population of follicles,leading to the premature depletion of follicle reserves.As expected,4-PBA,but not 3-MA,markedly ameliorated CDDP-induced follicle loss and preserved more healthy follicles by protecting primary and antral follicles from atresia.Consistent with this,a TUNEL assay showed that CDDP-induced granular cell apoptosis peaked on day 3(P<0.01),and was markedly ameliorated by 4-PBA(P<0.05)but not by 3-MA(P>0.05).By day 7,the intensity of granular cell apoptosis had decreased in the CDDP group,and 4-PBA slightly(but not significantly)reduced the number of apoptotic cells.Consistent with the histological results,hormone measurements with ELISAs showed that exposure to CDDP for 7 days clearly reduced the plasma E2 content of the ovaries,and especially,increased the FSH level(both P<0.01),which is considered the most important biochemical indicator of clinical POI.The 4-PBA treatment clearly inhibited the CDDP-induced increase in FSH but not the reduction in E2,whereas the 3-MA treatment had negligible effects on these changes.Furthermore,an immunoblotting analysis of whole-mount ovarian proteins showed that in vivo treatment with 4-PBA blocked the increases in the protein levels of ATG12,HSPA5,HSP90AB1,and cleaved PARP during CDDP treatment.The CDDP-induced increase in cleaved PARP was resistant to 3-MA treatment,although 3-MA markedly reduced the protein levels of ATG12.These results together provided strong in vivo evidence supporting the notion that the alleviation of ERS attenuated CDDP-induced granular cell death and ovarian damage.In summary,cisplatin can destroy ovarian follicles in the development of its mechanism may be related to cisplatin-induced ovarian endoplasmic reticulum stress.Endoplasmic reticulum stress can stimulate cell autophagy,excessive or sustained endoplasmic reticulum stress and autophagy eventually lead to apoptosis.In the body of the ovary,the main manifestations of atopic follicles increased,healthy mature follicles decreased,accompanied by increased apoptosis of granulosa cells,FSH increased,E2 decreased premature ovarian failure and other phenomena.Conclusion1.Cisplatin can reduce the expression of endoplasmic reticulum stress-related genes Hspa5 and Hsp90ab1 in mice;2.HSP90AB1 and HSPA5 play an important role in granulosa cells under the stimulation of cisplatin,knockdown can reduce the levels of endoplasmic reticulum stress,autophagy and apoptosis;3.Endoplasmic reticulum stress inhibitor 4-PBA can reduce the level of endoplasmic reticulum stress,autophagy and apoptosis induced by cisplatin in granulosa cells;4.Autophagy inhibitor 3-MA can reduce cisplatin-induced granulosa cell autophagy levels,but no significant effect on endoplasmic reticulum stress and apoptosis;5.4-PBA can relieve cisplatin-induced ovarian damage. |
PDF Full Text Request