Study On The Protective Effect Of N-Benzyl DHA On Cyclophosphamide-Induced Ovarian Injury In Mice

Commonly used chemotherapy drugs may cause ovarian damage.Although hormone therapy has been used clinically to reduce the risk of ovarian damage,it is difficult to achieve the desired therapeutic effect and has significant side effects.Therefore,there is a need to develop new treatments and drugs for ovarian damage after chemotherapy.N-Benzyl DHA(N-benzyl docosahexaenamide,NB-DHA)is the first DHA derivative discovered and identified and synthesized from the plant maca by our group.Based on existing studies on DHA derivatives and ovarian diseases,we hypothesize that NB-DHA may have a more positive therapeutic effect on ovarian damage compared to DHA molecules.Objective:Based on the synthesis and purification of NB-DHA,we investigated the drug treatment potential of NB-DHA in mice with cyclophosphamide(CTX)-induced ovarian injury and the difference in efficacy compared with DHA-EE,and explored the related mechanism of action to provide new ideas and references for the treatment and drug development of ovarian injury.Methods:1.Synthesis and Purity Identification of NB-DHA and DHA-EEUsing fish oil as raw material,DHA-EE was synthesized by esterification method,and NB-DHA was synthesized by carbodiimide condensation method.Based on the previous research methods of the research group,high-performance liquid chromatography was used to separate and identify the purity of NB-DHA and DHA-EE,and the structures of NB-DHA and DHA-EE were preliminarily identified by GC-MS and infrared spectroscopy.2.The effect of NB-DHA and DHA-EE on CTX-induced ovarian injury in miceCyclophosphamide(CTX)was used as an inducer of ovarian injury model in mice.After gavage administration via DHA-EE and NB-DHA,blood and tissue samples were used to analyze motility cycle,ovarian morphology,follicle number,sex hormone levels,m RNA levels of relevant genes,granulosa cell apoptosis rate and immune cell levels.3.In vivo and in vitro catabolism of DHA and its co-crystal preparationBased on the previous studies on NB-DHA in animal experiments,in order to investigate the decomposition of NB-DHA and the main metabolic processes and absorption pathways in vivo,we simulated the establishment of gastric and intestinal fluid environment in vitro to study the decomposition of NB-DHA;analyzed the composition and content of each fatty acid in serum at different time points after in vivo administration in rats to explore the metabolism of NB-DHA in vivo.The catabolic products were analyzed using gas chromatography and gas chromatography-mass spectrometry techniques.NB-DHA was modified into co-crystals,and the preparation effect and solubility improvement effect of co-crystals were initially explored.Results:1.Synthesis and Purity Identification of NB-DHA and DHA-EEFree fatty acids were obtained by hydrolysis of fish oil by lipase.NB-DHA was synthesized from free fatty acid and benzylamine,and DHA-EE was synthesized from free fatty acid and ethanol.98.3%purity of NB-DHA monomer compound and 96.2%purity of DHA-EE monomer compound were obtained by high performance liquid chromatography analysis.It was identified by infrared spectroscopy and gas chromatography analysis that NB-DHA has a benzene ring and amide bond structure,and DHA-EE has an ester bond structure.2.The alleviation effect of NB-DHA and DHA-EE on CTX-induced ovarian injury in miceBoth NB-DHA and DHA-EE can improve estrous cycle disturbance,the proportion of normal follicles and atretic follicles,hormone levels,regulate the expression of ovarian-related genes,reduce granulosa cell apoptosis,increase the expression of anti-Müllerian hormone and follicle-stimulation hormone receptors in ovarian tissue that increase the ratio of CD4~+/CD8~+.NB-DHA improved ovarian injury better than DHA-EE.3.In vitro and in vivo catabolism results of DHA and preparation of co-crystalsThe results of in vitro decomposition experiments showed that the decomposition rate of DHA-EE in intestinal fluid increased sharply,and the decomposition rate of NB-DHA in gastric fluid increased sharply.The in vivo metabolism results showed that DHA-EE was metabolized to hydroxyperoxy-docosahexaenoic acid and absorbed after 4 h of administration.N-eicosane and tetracosanol were detected in serum 2 h after NB-DHA administration.NB-DHA is absorbed into the blood faster than DHA-EE.Try to prepare NB-DHA vanillic acid co-crystal,the solubility of NB-DHA vanillic acid co-crystal is between NB-DHA and vanillic acid.Conclusion:In this study,two kinds of DHA were synthesized,namely DHA-EE and NB-DHA,and the purity was more than 95%.To further study the relieving effects of two DHAs on ovarian injury in mice,from changes in body weight,follicle number at all levels,sex hormone levels,expression of ovarian development-related genes,damage to ovarian granulosa cells,expression of AMH and FSHR in follicles and It was concluded from the aspects of immune cell typing in blood that high dose NB-DHA had the best remission effect,and under the same dose condition,the effect of NB-DHA was better than DHA-EE.Both DHA-EE and NB-DHA ameliorated ovarian damage in mice through multiple epigenetic mechanisms.Therefore,NB-DHA is a promising drug for the treatment of ovarian injury.Combined with the results of in vitro and in vivo metabolism studies,the mode of action of DHA-EE in vivo is that it is absorbed in the small intestine after converting DHA to hydroxyperoxy-docosahexaenoic acid by pancreatic lipase.NB-DHA acts by pepsin in vivo,and n-eicosan and tetracosanol can be detected in serum 2 h after administration.NB-DHA is absorbed into the blood at a faster rate than DHA-EE.The preparation of NB-DHA vanillic acid co-crystal could improve the solubility of NB-DHA and provide a theoretical basis for the subsequent multi-dosage transformation of NB-DHA.