| Part Ⅰ Effect of Transcutaneous Acupoint Electrical Stimulation on Protecting Against Radiotherapy-induced Ovarian DamageObjective:Transcutaneous electrical acupoint stimulation(TEAS)is a form of physical therapy,which uses self-adhesive electrode tablets placed on the surface of acupoints and affects the functions of tissues and organs by a certain amplitude of electric current through neurohumoral action.Although TEAS has been widely used in the treatment of ovarian dysfunction for a long time,the mechanism is not clear.In the present study,we aimed to explore the protective effect of TEAS on radiotherapy-induced ovarian damage.Methods:A total of sixty 8-week-old C57BL6 female mice were randomly divided into 6 groups with 10 mice in each group:①control without TEAS treatment(control-),②control with treatment(control+),③irradiation without early TEAS treatment(IR 2D-),④irradiation with early treatment(IR 2D+,TEAS treatment initiated at 2 days after irradiation),⑤irradiation without late TEAS treatment(IR1W-),and⑥ irradiation with late treatment(IR 1W+,TEAS treatment initiated at 1weeks after irradiation).The radiotherapy model mice received whole-body X-ray irradiation at a single dose of 4 Gy,and the control mice received whole-body X-ray irradiation at a single dose of 0 Gy.TEAS stimulation lasted for 30 minutes with continuous wave of a low frequency of 2 Hz(200 μs pulse width)and the intensity of 1 mA.The treatment was given once a day for 6 consecutive days,over a 2-week period.The skin around the acupoints shivering mildly indicated the proper dose.The analogue TEAS treatment group was only given acupoint application of electrode tablets,but received no electrical stimulation.During the treatment,mice daily for the general condition and the estrous cycle.After the treatment,enzyme-linked immunosorbent assay(ELISA)was applied to test the serum levels of anti-mullerian hormone(AMH).The ovarian tissue sample was processed with hematoxylin eosin(HE)staining as well as the number of ovarian follicles were counted.Results:Compared with control-group,mice receiving irradiation alone(both IR2D-and IR 1W-mice)showed disordered estrous and lower serum level of AMH(3.03±0.27 ng/ml vs.5.17±0.44 ng/ml,P=0.014;2.30±0.18 ng/ml vs.5.17±0.44ng/ml,P=0.003;respectively).Consistent with these data,the ovarian sizes and weight significantly decreased in the IR 2D-and IR 1W-groups(P<0.001).The numbers of primordial(74.00±14.21 vs.576.00±42.79,P<0.001),primary(47.00±5.12 vs.342.50±31.81,P<0.001)and secondary(16.40±2.39 vs.41.60±6.76,P=0.002)follicles were significantly decreased in the IR 2D-group than that in the control-group.Whereas there was no difference in the number of antral follicles between the IR 2D-group and the control-group(P=0.637).Moreover,all stages of follicle counts were dramatically reduced further in IR 1W-group.The histopathological examination showed that the morphology of the ovaries was obviously impacted by irradiation.In the IR 2D-group,the oocytes shrunk to a small size or completely disappeared in the later stages of atresia.Most of the primary follicles had only a single layer of cuboidal granulosa cells.Moreover,in the IR 1Wgroup,the number of follicles with atresic features was higher than that in the IR 2D group.Meanwhile,the degenerating follicles showed that granulosa cells exhibited separation and loss of integrity.In addition,TEAS treatment initiated at 2 days after irradiation(IR 2D+)successfully restored estrous cycle,significantly increased the serum level of AMH(4.13±0.18 ng/ml vs.3.03±0.27 ng/ml,P=0.028)and the number of primordial follicle(189.50±33.64 vs.74.00±10.21,P=0.006),whereas there was no statistically difference in the count of primary,secondary and antral follicles between the IR 2D+and IR 2D-groups(P>0.05).However,irradiation with late TEAS group(IR 1W+)had no protective effects on ovarian function.Conclusions:Whole body single 4Gy X-ray irradiation could induce ovarian damage,including estrous cycle disorder,AMH level decrease,and ovarian morphology damage and all stages of follicles counts loss.Moreover,TEAS treatment initiated at 2 days after irradiation had protective effects on radiation-related ovarian damage,and treatment started early after irradiation may be of more effective.Part Ⅱ Study on the Mechanism of Transcutaneous Acupoint Electrical Stimulation Protecting Ovarian Function in Radiotherapy Mice Objective: Irradiation could induce ovarian damage,including estrous cycle disorder,AMH level decrease,and ovarian morphology damage and all stages of follicles counts loss.Moreover,TEAS treatment after irradiation had protective effects on radiation-related ovarian damage.However,the possible mechanisms are poorly elucidated.PTEN/Akt/FOXO3 a signaling pathway is one of the important molecular mechanisms for the maintenance,activation,growth and survival of primordial follicle,and which is an important transcription factor associated with the oxidative stress response.Exposure to X ray can result in significant damage to organs through generating reactive oxygen species(ROS).This imbalance between pro-oxidants and antioxidants would damage mitochondrial function,leading to granulosa cell and follicular apoptosis.The purpose of this part was to elucidate the possiblemechanisms of TEAS on the protection of ovarian function.Methods: A total of forty 8-week-old C57BL6 female mice were randomly divided into four groups with 10 mice in each group: ?control without TEAS treatment(control-),②control with treatment(control.),③irradiation without early TEAS treatment(IR 2D-),?irradiation with early treatment(IR 2D+,TEAS treatment initiated at 2 days after irradiation).The radiotherapy model mice received whole-body X-ray irradiation at a single dose of 4 Gy,and the control mice received whole-body X-ray irradiation at a single dose of 0 Gy.TEAS stimulation lasted for30 minutes with continuous wave of a low frequency of 2 Hz(200 ps pulse width)and the intensity of 1 mA? and the treatment was given once a day for 6 consecutive days,over a 2-week period.The skin around the acupoints shivering mildly indicated the proper dose.The analogue TEAS treatment group was only given acupoint application of electrode tablets,but received no electrical stimulation.The expression levels of phosphorylated protein and total protein of PTEN,Akt,F0X03 a,GSK3p and ERK1/2 were quantified by western blot.Immunofluorescence analysis was used to detect phosphorylated FOX03 a expression and nuclear export of phosphorylated F0X03 a in primordial follicle using anti-LHX8 and anti-phosphorylated F0X03 a.Besides,the superoxide dismutase 2(SOD2),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)contents in ovarian tissue were measured by the method of ELISA,colorimetry and thiobarbituric acid reaction(TBA)respectively.Results: Western blotting showed increased phosphorylation and the relative level of PTEN,Akt,GSK3 p and F0X03 a in the irradiation alone treatment group(IR2D-),whereas TEAS significantly attenuated the level of irradiation-induced PTEN,Akt,GSK3 P and F0X03 a phosphorylation.As we known,F0X03 a is phosphorylated and exported from the nucleus during primordial follicle activation, the cytosolic localization of phosphorylated F0X03 a is an indicator of primordial follicle activation.Immunofluorescence analysis showed that F0X03 a was highly expressed in primordial follicles of all groups.Whereas,phosphorylated F0X03 a was dramatically increased and exported from nuclei of primordial follicle in the IR2D-mice,and TEAS treatment successfiilly inhibited irradiation-induced of F0X03 a shuttling from nucleus to the cytoplasm.Consistent with these data,the irradiation alone group(IR 2D-)showed a significant reduction of contents of SOD2 and GSH-Px and increase of MDA in ovarian tissue homogenate.Furthermore,TEAS significantly increased the levels of SOD2 and GSH-Px and decreased the level of MDA,leading to improve the antioxidant capacity of ovarian tissue.Conclusions: The whole body single 4Gy X-ray irradiation significantly depleted ovarian primordial follicle pool by activating PTEN/Akt/F0X03 a pathway in mice.TEAS treatment successfiilly inhibited irradiation-induced activation of PTEN/Akt/F0X03 a pathway and phoshphorylation F0X03 a shuttling from nucleus to the cytoplasm,which attenuate overactivation of primordial follicle.Furthermore,TEAS significantly increased the levels of SOD2 and GSH-Px,and decreased the level of MDA,in order to improve the antioxidant capacity of ovarian tissue.These results suggest that TEAS prevents in*adiation_induced primordial follicle activati^and improve the antioxidant capacity of ovarian tissue via suppression of F0X03 a phosphorylation and nuclear export. |
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