An In Vitro Mechanistic Study Of Doxorubicin-induced Toxicity In Ovarian Follicles Based On 3D Microfluidics And FRET Technologies

Doxorubicin(DOX)is commonly used as an effective antineoplastic drug.However,the clinical application of DOX is limited due to its harmful or even irreversible side effects on the ovaries,which frequently lead to severe gynecological complications.Once the functional unit of the ovary,the ovarian follicle(OF),is damaged,it cannot regenerate.Although several researchers have tried multiple techniques to study the DOX-induced toxic effects on OFs,its molecular mechanism of toxicity is still unclear.Because the cellular functions of OFs mainly depend upon certain crucial signaling proteins and the activity of these proteins alters at different stages of the folliculogenesis,it is hard to monitor their alteration with the disturbance of neurological and humoral factors in vivo,In order to further explore the molecular mechanism of DOX-induced toxicity,two key issues are needed to be addressed.Firstly,a research platform is necessary to be developed to simulate the physiological microenvironment and control the dynamic growth of OFs in vitro.Secondly,some genetically engineered molecular probes are required to monitor the dynamic changes in the activity of specific signaling proteins during the folliculogenesis.Therefore,a new microfluidic chip was developed to study the role of different signaling molecules in DOX-induced toxic effects on 3D-cultured OFs in vitro by measuring OFs diameter,their hormone secretions,and mRNA levels of apoptosis related genes.Moreover,the Ca2+source of DOX-induced intracellular Ca2+concentration([Ca2+]i)increase and the mechanism of DOX-induced toxicity in OFs via Src/Ca2+/PIM signaling pathway were revealed using fluorescence resonance energy transfer(FRET)technology.The detailed contents of the research work are as follows:1.A novel microfluidic chip was proposed and established to simulate the in vivo like microenvironment of the OFs.In this study,for culturing a single OF in the ovary-like microenvironment in vitro,a three-dimensional culture microfluidic chip containing five inlets,one outlet,and a circular culture chamber was fabricated with polydimethylsiloxane.20 intact human OFs(160-220 μm)were isolated from ovarian tissues and encapsulated in the three-dimensional calcium alginate hydrogel beads before culturing them on the chip.For the verification of proper functioning of the chip,the diameter and 17β-estradiol and testosterone secretions of the single human OF cultured on the chip were compared to that of the OF cultured in the dish under the same conditions for eight days.The results showed that the structure of OFs cultured on the chip was intact,and their growth and hormone secretion were normal,which confirmed that the chip supported the normal growth of an OF and the dynamic study of its cellular functions.Therefore,this chip was used in the following research for studying the toxic effects of DOX in OFs.2.The role of different signaling molecules in the mechanism of DOX-induced OFs toxicity was demonstrated.The rat OFs(160-220 μm)were isolated mechanically and cultured on the microfluidic chip mentioned above in this study.The on-chip cultured OF was treated with DOX(200 nM)and the inhibitors of Src kinase,Ca2+,and proviral-integration site for Moloney-murine leukemia virus(PIM)kinases(PP1,2APB,and AZD1208.respectively).Individual and combined effects of these drugs were studied on OFs growth and 17β-estradiol secretion.Besides,the RNA levels of beta 1,4-galactosyltransferase,polypeptide 2(B4GALT2),and unc5 homolog C(UNC5C)genes of DOX-exposed OFs were detected by RT-qPCR,and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)experiments were also conducted to check the DOX-induced OFs apoptosis.The results showed that the DOX application reduced the OFs growth and hormone secretion and induced apoptosis in the OFs.Moreover,DOX-induced toxic effects were enhanced by the Src and PIM inhibitors,while reduced by the Ca2+channel inhibitor.These findings verified the DOX-induced apoptosis in OFs and suggested the involvement of corresponding signaling proteins in the toxic mechanism of DOX in OFs.3.The changes in DOX-induced[Ca2+]i and its molecular mechanism in OFs were identified,FRET-base Cyto-Ca2+ and ER-Ca2+ biosensors were used to test the real-time Ca2+concentration in the cytosol and endoplasmic reticulum(ER),respectively.In addition,FRET-based Src biosensor was used to reveal the role of dynamic Src activity in DOX-induced[Ca2+]i alteration.The rat OFs(150-180μm)transfected with these biosensors were pretreated with PP1,2APB,and EGTA to inhibit the Src activity,ER-Ca2+release,and extracellular Ca2+ influx,respectively.The results showed that DOX up-regulated the[Ca2+]i smoothly,which could be inhibited by PP1 and 2APB,indicating that DOX increased the[Ca2+]i mainly through the ER-Ca2+ release in the Src kinase activity-dependent manner,but not from the extracellular Ca2+influx.4.A new genetically engineered FRET-based PIM biosensor,named EPHY,was developed for studying the role of PIM kinases in the mechanism of DOX-induced toxicity.The biosensor contained the PIM1 sequence,substrate HSP90,and a pair of ECFP and Ypet fluorescent proteins to monitor the real-time PIM activity in live cells.For verifying the validity and specificity of EPHY biosensor,different concentrations of PIM inhibitor AZD1208(0.5,1,and 1.5 nM)were applied on EPHY-transfected HeLa cells.The decrease in the FRET ratio was observed with the increase in AZD1208 concentration,confirming that EPHY could be used for detecting the real-time changes in the activity of PIM kinase.To identify the role of Src and Ca2+in DOX-induced activity of PIM kinase,EPHY-transfected rat OFs were pretreated with PP1 and 2APB,respectively.The results demonstrated that the DOX-induced increase in PIM activity was attenuated by these drugs,which revealed that Src could regulate PIM activity mainly via ER-Ca2+release in OFs on DOX application.In conclusion,this study develops a newly designed microfluidic chip and a novel FRET-based biosensor.The chip can provide an ovary-like microenvironment to the OFs cultured in vitro,which can be used to monitor the alteration in their growth and fluctuations in their hormone secretion in response to different drugs.Besides,the genetically constructed FRET-based biosensor can monitor the real-time PIM activity in the live cells cultured in vitro.Based on the research using these novel tools,this study indicates that Src/Ca2+/PIM pathway can be intervened to shield the female endocrine and reproductive systems against the DOX toxic effects.In addition,this research presents a novel application of a combination of microfluidics and FRET technologies,which provides a new method to the researchers for conducting several studies related to the pharmaceutical screening,signal transduction,and molecular toxicology.