| Objective:With the increasing plastic emissions,the problem of global plastic pollution continues to worsen,and the impact of plastic particles on human health has gradually attracted scholars’ attention.Plastic particles with a diameter of less than 100 nm are called nano plastics(NPs).They may be more toxic than large plastic waste and microplastics due to their higher concentration and small size.It has been reported that NPs can induce oxidative stress and cause damage to the female reproductive function of aquatic animals,but the research on female mammals remains unclear,and the underlying mechanism is still unknown.The Hippo pathway has been confirmed to participate in cell damage caused by oxidative stress,and studies conducted by our team and researchers have shown that it plays an essential role in regulating ovarian function.Previous studies have suggested that NPs may regulate cellular oxidative stress through the Hippo signaling pathway,thus affecting ovarian function.Therefore,this study explored the effects of NPs exposure on ovarian function through female mice and human ovarian granulosa cells(GCs),and further explored its regulatory mechanism on the Hippo signal pathway.Moreover,we also investigated the intervention effect of Chinese patent medicine extracts(salidroside)with antioxidant activity on NPs toxicity,to provide a new idea for the development and application of traditional Chinese medicine and a new treatment strategy for patients with environmental damage to ovarian function.Methods:1.The 5-week-old healthy balb/c female mice were randomly selected to receive 1mg of fluorescent polystyrene nanoplastics(FPS-NPs)orally for two consecutive days.After the ovarian tissue was collected,frozen sections were prepared,and confocal microscopy was performed to detect the accumulation of FPS-NPs in the ovary.2.Healthy balb/c female mice at 5 weeks of age were randomly assigned to two groups.One group received orally 1mg of polystyrene nanoplastics(PS-NPs)daily for 5 weeks,while the other group received the same volume of ultra-pure water.Then,one mouse from the control group and one from the PS-NPs treatment group were paired with a healthy male breeding mouse.After 3-4 weeks,the number of successful pregnancies and offspring was recorded.Serum and ovarian samples were collected and the ovarian tissue structure was observed using HE staining to classify and count the follicles.The ovarian reserve of mice was evaluated by detecting the level of anti-Mullerian hormone(AMH)in the serum using ELISA.3.The level and distribution of apoptosis in ovarian tissue were determined using the TUNEL method.The expression level of apoptosis-related proteins(bax/bcl2)was measured by Western blot to investigate the potential mechanism of ovarian injury.4.This study used in vitro cultured human ovarian granulosa cell line(KGN).Firstly,immunofluorescence staining with Cy3-labeled phalloidin and DAPI was performed to observe the internalization of PS-NPs by KGN cells.Secondly,KGN cells were treated with PS-NPs at different concentrations(50 μg/ml,100 μg/ml,and 200 μg/ml)to observe changes in cell morphology and viability,and to select the appropriate exposure concentration of PS-NPs.Thirdly,the EDU and Annexin & V assays were conducted to measure the proliferation and apoptosis levels,respectively,to evaluate the damaging effects of PS-NPs on KGN cells.Finally,the ROS levels induced by PS-NPs were assessed using the DCFH-DA probe.5.KGN cell lines were cultured in vitro and exposed to 100 μg/ml PS-NPs for 2 hours to extract proteins from KGN cells.The expression levels of major components of the Hippo signaling pathway(MST1,P-MST1,LATS1,P-LATS1,YAP1,and PYAP1)were determined by Western blot to explore the effect of PS-NPs on the Hippo signaling pathway.6.At concentrations of 100 μM and 200 μM,the morphology of KGN cells treated with salidroside did not show significant improvement,while at 300 μM the abnormal morphology of KGN cells was significantly reduced and at 400 μM,the morphology of KGN cells was further improved.Compared with the PS-NPs group,the salidroside treatment group showed a significant decrease in intracellular ROS levels in KGN cells(P<0.05)and alleviated the impact of PS-NPs on NOX4 and GPX1 in KGN cells(P<0.001).Salidroside can inhibit PS-NPs-induced oxidative stress in KGN cells.Results of EDU and Annexin&Ⅴ assays showed that the proliferation level of KGN cells in the salidroside treatment group decreased while the apoptosis level increased(P<0.001).7.After the salidroside intervention,the major components of the Hippo signaling pathway were detected by Western blot to further explore the regulatory mechanism of PS NPs on the Hippo signaling pathway in KGN cells.Results:1.FPS-NPs ingested orally can enter the ovary and accumulate among granulosa cells around the follicle,but rarely enter the oocyte.2.Compared with the control group,the successful pregnancy rate of the PSNPs group decreased significantly,suggesting that fertility was impaired.However,there was no significant difference in the time to pregnancy and the litter rate of pregnant mice between the control group and the PS-NPs exposure group.The number of healthy follicles in the PS-NPs group decreased significantly(P<0.01),and the level of AMH in the serum of mice decreased significantly(P<0.01).There was no significant difference in ovarian weight and ovarian organ coefficient(P>0.05).3.TUNEL-positive cells in the ovary of the PS-NPs group increased significantly and were mainly concentrated in granulosa cells around the follicle.In addition,the apoptosis-related gene Bax was significantly up-regulated in the ovarian tissue of mice exposed to PS-NPs(P<0.01),while Bcl2 was significantly downregulated(P<0.001).4.Fluorescence results showed that PS-NPs could penetrate the cell membrane and enter KGN cells,most of them accumulated around the nucleus,and a small part could enter the nucleus.There is no significant difference in KGN cell morphology between the 50 μg/ml PS-NPs treatment group and the control group.However,exposure to 100 μg/ml of PS-NPs caused abnormal morphology of KGN cells,and 200 μg/ml PS-NP can cause KGN cell death.The CCK-8 results showed that 50 μg/ml PS-NPs had no significant effect on cell viability,100 μg/ml PS-NPs significantly reduced cell viability,while 200 μg/ml PS-NPs caused significant cell death.In the PS-NPs group,the proliferation level of KGN cells decreased significantly(P<0.001)and the apoptosis rate increased significantly(P<0.001).The content of ROS in the PS-NPs group increased significantly(P<0.001).In addition,oxidative stress-related gene NOX4 m RNA was significantly up-regulated(P<0.001),while GPX1 was significantly down-regulated(P<0.001).5.In the PS-NPs group,the expression of MST1(P-MST1),LATS1(P-LATS1)and P-YAP1 in KGN cells was significantly increased(P<0.001),while the total protein of YAP1 was not significantly different(P>0.05).6.Compared with the PS-NPs group,the ROS level of KGN cells in the Sal group was significantly reduced(P<0.05),and the effect of PS-NPs on NOX4 and GPX1 in KGN cells was also reduced(P<0.001).EDU and Annexin&Ⅴ results showed that the proliferation level of KGN cells decreased and apoptosis level increased in the salidroside treatment group(P<0.001).7.Salidroside significantly decreased MST1(P-MST1),LATS1(P-LATS1),and P-YAP1 up-regulated by PS-NPs(P<0.05),suggesting that Oxidative stress may be a major cause of Hippo pathway dysregulation.Conclusion:1.PS-NPs induce oxidative stress and Hippo pathway dysregulation,leading to granulosa cell apoptosis and reduced fertility in mammals.2.Salidroside is a potential therapeutic agent that can rescue PS-NPs-induced oxidative stress in granulosa cells and mitigate cell damage. |
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