The Protective Effects And Mechanisms Of Quercetin On Cisplatin-induced Ovarian Injuries In Mice

Background:With the popularization of early screening methods,changes in lifestyle and the increase of life pressure,the age at which cancer is diagnosed in China continues to be advanced.According to the2020 White Paper on Quality of Life in China,the proportion of young patients（15-39 years old）suffering from gastric cancer,cervical cancer,colorectal cancer,kidney cancer,and some other cancers is rising.At the same time,with social development and changes in fertility concepts,the average reproductive age of women in Cina has been slowly delayed,and late marriage and childbearing have become more and more popular.Therefore,the protection of ovarian endocrine and reproductive function in female patients with malignant tumors has attained more and more attention.Cisplatin,as a broad-spectrum anticancer drug,can inhibit the DNA replication and transcription of tumor cells and can be used for malignant tumors such as head and neck squamous cell carcinoma,lymphosarcoma,ovary cancer,and cervical cancer.However,its satisfactory anticancer effects are accompanied by toxic effects on normal organisms,such as nephrotoxicity,digestive tract toxicity,ototoxicity,cardiotoxicity,and ovarian toxicity.The ovarian toxicity of cisplatin can damage the ovarian endocrine function,leading to the disorder of female sex hormone secretion,the decrease of LH,E₂,and P levels,and an increase in FSH levels.Meanwhile,the decreased AMH level and the decreased number of follicles lead to decreased fertility ability or even infertility.In recent years,there has been increasing emphasis on the use of natural products such as flavonoids,especially quercetin,to treat diseases.As a natural compound,quercetin has traditionally been used to prevent or treat a variety of diseases including cancer,cardiovascular disease,polycystic ovary syndrome（PCOS）,obesity,chronic inflammation,and reproductive dysfunction.Studies have shown that quercetin can act as an anti-apoptotic agent and antioxidant to protect against various organ damage.In the last few years,the journal Nature Communication has published several articles revealing the significant role of quercetin in anti-aging and aging-related degenerative diseases.Researchers have revealed that quercetin and cisplatin have synergistic tumor suppressor effects in the treatment of ovarian cancer and cervical cancer in both vivo and in vitro experiments,but the effect of combined quercetin on cisplatin-induced damage to normal ovarian function is still unclear.Surgery and cisplatin-based neoadjuvant chemotherapy are the two main methods for fertility preservation in early-stage ovarian and cervical cancers.What is more,the damage of external toxins including chemotherapy drugs is also an important cause of premature ovarian insufficiency（POI）.How to reduce the damage caused by external toxins to the ovary and preserve the ovarian reserve has become an urgent problem for clinicians.Therefore,establishing a model of cisplatin-induced ovarian function impairment in female mice and exploring the protective mechanism of quercetin on cisplatin-induced ovarian function damage may show the theoretical significance and clinical guiding value for exploring the possibility of quercetin as an ovarian protectant when cisplatin is used in the treatment of gynecological cancer patients with fertility preservation requirements or when the ovaries are damaged by some other toxins.Methods:It has been reported that intraperitoneal injection of 1.5mg/kg of cisplatin in female mice for 15 days can lead to impaired ovarian function.This cumulative human equivalent dose will not cause infertility and is close to the commonly used chemotherapy dose of cisplatin in clinical practice（75 mg/m²）.Therefore,in this study,a cisplatin-induced ovarian injury model in female mice was established by intraperitoneal injection of cisplatin once a day for 15 days in 7-week-old female ICR mice.According to literature reports,100mg/kg of quercetin can protect the spinal cord,liver,lung,and some other tissue from damage.This experiment was divided into four groups:control group（normal saline）,quercetin（Que）group（100mg/kg）,cisplatin（Cis）group（1.5mg/kg）,combined drugs（Cis+Que）Group（quercetin 100mg/kg+cisplatin1.5mg/kg）.In the study,the protective effect and preliminary mechanism of quercetin on the ovary after cisplatin-induced damage to the ovary were discussed in 4 parts.Part I Quercetin reduced cisplatin-induced follicle loss:The general conditions of mice,such as body weight,ovarian weight,and food intake were observed to evaluate the effect of drugs on the health of mice;fluorescence Tunel and immunohistochemistry（IHC）of Cleaved-Caspase 3were used to evaluate the apoptosis of each group;IHC of PCNA protein was used to evaluate the proliferation of each group;the morphological changes of ovarian tissue were observed by HE staining of ovarian tissue;the changes in the number of follicles in each group were observed by follicle count.Part II Quercetin alleviated the damage of cisplatin on ovarian function in mice:the effects of drugs on ovarian endocrine function in mice were evaluated by observing the estrous cycle of mice and detecting the level of sex hormones;the effects of drugs on the reproductive function of mouse ovaries were evaluated by detecting AMH and mouse mating experiments.Part III Label-free quantitative detection of mouse ovarian proteins:We quantitatively detect mouse ovarian proteins through proteomics to find differential proteins and conduct bioinformatics analysis to preliminarily explore the effects of two drugs on the ovary;Part IV preliminary exploration of the mechanism of how quercetin alleviated cisplatin-induced ovarian damage:the oxidative stress level in mouse ovary was detected,and the apoptosis-related proteins（Cleaved-Caspase 3,Bax,Bcl-2）and the expression of primordial follicle activation-related proteins（p-PTEN/PTEN,p-Akt/Akt,p-m TOR/m TOR,p-Foxo3a/Foxo3a,p-rp S6/rp S6）were detected by western blotting（WB）to evaluate the effect of drugs on apoptosis and PI3K/PTEN/Akt follicle activation signaling pathway in each group.Results:（1）Compared with the mice in the Control group,the mice in the Cis group had less food intake,body weight,and ovarian weight.The combined use of quercetin reduced the toxicity of cisplatin,and the food intake,body weight,and ovarian weight of mice increased;（2）The apoptosis of ovarian granulosa cells,early follicle activation,and proliferation in the Cis group were increased compared with those in the Control group,and quercetin could reduce cisplatin-induced granulosa cell apoptosis and early follicular activation;（3）Cisplatin reduced the number of primordial follicles,decreased ovarian reserve,increased primary follicles,and decreased antral follicles in mice,and both primordial follicles and antral follicles in the Cis+Que group increased compared with those in the Cis group;（4）The estrous cycle of the mice in the Cis group was stagnant in the diestrus,the serum estrogen level decreased,and the secretion of follicle-stimulating hormone increased.The combined use of quercetin increased the estrous frequency of the mice,increased the serum estrogen level,and decreased the FSH level;（5）In the first round of mating rounds,the birth spacing time of mice in the Cis group was longer than that of the Control group,but there was no statistical difference in the second and third rounds.The expression level of anti-Müllerian hormone in female mice in the Cis group decreased,and the litter size decreased,while the serum anti-Müllerian hormone level and litter size in the Cis+Que group were higher than those in the Cis group;（6）The differential proteins between the Cis group and the Control group were enriched in the response to reactive oxygen species,the regulation of reactive oxygen species metabolism,the metabolic process of small molecules,the metabolic process,the extracellular structure and organization,the development process,biological adhesion,cell proliferation,biological adhesion,and some other biological processes;after combined treatment with quercetin,the differential proteins can be enriched in the process of antioxidant activity,and quantitative protein detection also found that the apoptotic protein in the Cis+Que group was lower than that in the Cis group,while the anti-apoptotic protein was significantly higher than that in the Cis group.Compared with the Cis group,the level of PTEN,an important negative regulator in the PI3K/AKT signaling pathway,was increased;（7）Quercetin increased the expression level of anti-oxidative stress genes in the mouse ovary,decreased the level of cisplatin-induced ovarian apoptotic protein,increased the expression of anti-apoptotic protein,and reduced cisplatin-induced phosphorylation of key proteins related to follicle activation in PI3K/PTEN/AKT signaling pathways.Conclusion:（1）In this study,an animal experimental model was constructed,and quercetin alleviated cisplatin-induced ovarian damage in mice;（2）Quercetin attenuates cisplatin-induced apoptosis,reduces the loss of primordial follicles and antral follicles,and increases ovarian reserve function;（3）Quercetin improves the ovarian endocrine function and reproductive function of mice damaged by cisplatin;（4）Quantitative protein detection suggested that quercetin may play a role in regulating oxidative stress,ovarian cell apoptosis,and PI3K/AKT pathway in the ovary;（5）The results of PCR and WB verification showed that quercetin could increase the anti-oxidative stress ability of the ovary,reduce the expression of apoptosis proteins in ovarian cells,increase the expression level of anti-apoptotic proteins and inhibit the phosphorylation of key proteins related to follicle activation in the PI3K/PTEN/Akt signaling pathway to attenuate cisplatin-induced ovarian toxicity.