Role And Mechanism Of Mitophagy In Cerebellar Purkinje Cell Dendrite Damages Caused By Nonylphenol Exposure

Objective:Nonylphenol is a persistent organic pollutant that can be enriched through the food chain and enter the human body,posing a threat to human health.More and more evidence has shown that nonylphenol can cause a variety of body damage,especially irreversible damage to the nervous system.Mitophagy is a key mechanism of mitochondrial quality control and plays an important role in maintaining nervous system homeostasis.Recent studies have shown that nonylphenol can cause damage to the neural development and even cognitive behavior of progeny when it crosses the blood-brain barrier.However,studies on whether perinatal exposure to nonylphenol can cause dendrite damages of neurons in cerebellum have not been carried out yet.In this study,we established a model of nonylphenol exposure in Perinatal rats to investigate the effect of mitophagy on the dendrites of nonylphenol in the cerebellar Purkinje cells of offspring rats and its possible mechanism.Based on this,a nonylphenol exposure model of SH-SY5Y human neuroblastoma cells was established,the role and mechanism of mitophagy in the dendrite damages of offspring cerebellum Purkinje cells induced by nonylphenol exposure during pregnancy were investigated by inhibiting the classical mitophagy pathway PINK1/Parkin.Methods:1.Established maternal nonylphenol exposure model during pregnancy and feeding period:SPF grade adult female and male SD rats（260-280 g）were purchased from the Animal Experimental Center of China Medical University.After one week of adaptive feeding,the rats were put in the same cage（1:1 male/female ratio）,and vaginal plug was examined the next morning.If vaginal plug was found,it was recorded as the Gestational Day 0.Pregnant rats were fed separately and randomly divided into 0,2,10 and 50mg/kg/day Nonylphenol exposure groups according to body weight,and the 0 mg/kg/day group was the control group.From Gestational Day 0 to Postnatal Day 21,different doses of Nonylphenol were dissolved in corn oil,and female rats were given gavage,the intragastric volume was 0.5ml/100 g bw.Each group consisted of 16-18 pregnant mice.The offspring rats were weaned at 21 days after birth.In order to avoid the interference of sex hormones in the experiment,only the offspring male rats were selected as research objects.2.Establishment and induction of nonylphenol exposure model of nerve cell:Human neuroblastoma cells（SH-SY5Y）were infected with nonylphenol at a concentration of30μM,while the control group was treated with nonylphenol for 24 hours.At the same time,SH-SY5Y cells were induced by retinoic acid with a final concentration of 10μM,and cells were collected for subsequent experiments.Neurites may be induced by retinoic acid.After the above treatment,cell samples were collected for testing.3.Detection of dendrite damages in cerebellar Purkinje cells:Paraffin sections of rat cerebellar tissues were made,and Calbindin D-28K immunofluorescence staining was performed to observe the dendrite damages in the length and branch complexity of cerebellar Purkinje cell dendrites,and the typical planar morphological model of Purkinje cell dendrites was drawn.Image J was used for quantitative analysis of the total length of dendrites and the number of branch intersections.4.Detection of related indicators of excessive activation of cerebellar mitochondria in offspring rats:The contents of mitochondrial marker proteins TOM23 and TIM23,autophagy related protein LC3Ⅱ/Ⅰand P62 were detected by Western blot.The content of TIM23 protein and LC3 protein in offspring rat cerebellum were observed by immunofluorescence.5.Detection of related indexes of PINK1/Parkin pathway:Collected cerebellum tissues of offspring rats,and extracted mitochondria and cytoplasmic proteins from cerebellum tissues of offspring rats with tissue mitochondrial separation kit.The contents of PINK1 and Parkin in mitochondrial protein and cytoplasmic protein extracted from the offspring cerebellum were detected by Western blot6.Si RNA transfection of SH-SY5Y cell:In the pre-experiment,fluorescent-labeled CY3 si RNA was transfected to help optimize the transfection conditions of cell lines.Five concentration gradients were set for transfection of si RNA,respectively 0、30、50、70、90 and 100 n M.The transfected cells were cultured at 37℃in 5%CO₂ incubator,and the culture was changed 6 hours later.The transfection efficiency was observed by fluorescence microscope 24 h later.7.The detection of PINK1 knockdown alleviates the morphological injury of SH-SY5Y cell neurites induced by nonylphenol exposure:The morphological injury of SH-SY5Y cell neurites were observed under an optical microscope;The cells were stained with crystal violet and the morphological injury of the neurites were observed under an optical microscope.To investigate whether the transfection of PINK1 si RNA can alleviate the morphological injury of neurite.8.PINK1 knockdown alleviates the over-activation of mitochondrial autophagy in SH-SY5Y cells induced by nonylphenol exposure:immunofluorescence assay and laser confocal assay were used to observe the contents of mitochondrial autophagy related TOM20 protein,TIM23 protein,LC3 protein and P62 protein,and Western blot assay was used to detect the expression level of TIM23 and P62 protein.To explore whether the transfection of PINK1 si RNA can alleviate the overactivation of mitochondrial autophagy.Results:1.Nonylphenol exposure induced dendrite morphological injury in rats’bellar Purkinje cells:Calbindin D-28K immunofluorescence staining showed that,compared with the control group,2 mg/kg,10 mg/kg and 50 mg/kg groups decreased the intensity of dendrite fluorescence and shortened the total length of the Purkinje cells（P<0.05）.Sholl analysis showed that compared with the control group,the number of dendritic branch junctions and complexity of dendritic branches in 2 mg/kg,10 mg/kg and 50 mg/kg groups were decreased（P<0.05）.The above results showed a dose-dependent trend.2.Nonylphenol exposure during pregnancy and lactation caused excessive activation of mitophagy in offspring rats:Western blot results showed that the 2,10,and 50 mg/kg dose groups had a significant reduction in the content of mitochondrial marker protein TOM20 in the cerebellum of offspring rats compared with the control group（P<0.05）.Compared with the control group,the 10 mg/kg and 50 mg/kg dose groups had a significant reduction in the content of mitochondrial marker protein TIM23（P<0.05）.The 50 mg/kg dose group had a significant increase in the content of autophagy marker protein LC3Ⅱ/Ⅰin the cerebellum of offspring rats（P<0.05）.The 2 mg/kg dose group,10 mg/kg dose group,and 50 mg/kg dose group had a significant reduction in the content of autophagy marker protein P62 in the cerebellum of offspring rats compared with the control group（P<0.05）,and the changes in the content of the above proteins showed a certain correlation trend with the exposure dose.The results of immunofluorescence showed that with the increase of exposure dose of nonylphenol in the pregnancy and lactation group,the content of TIM23 protein in the cerebellum of offspring rats gradually decreased compared with the control group,and the content of LC3 protein gradually increased compared with the control group3.Nonylphenol exposure during pregnancy and lactation leads to activation of PINK1/Parkin pathway in the cerebellum of offspring rats:Western blot results showed that PINK1 protein levels in the mitochondria of the 2,10 and 50 mg/kg dose groups were significantly increased compared with the control group（P<0.05）.The 50 mg/kg dose group had a significant reduction in the content of PINK1 protein in the cytoplasm（P<0.05）.The 50 mg/kg dose group had a significant increase in the content of Parkin in the mitochondria（P<0.05）,and the 10 mg/kg and 50 mg/kg dose groups had a significant reduction in the content of Parkin in the cytoplasm（P<0.05）.Both PINK1 and Parkin proteins were transferred from the cytoplasm to the mitochondria,and PINK1/Parkin pathway was activated.4.SH-SY5Y cell model with PINK1 knockdown was established successfully:The results of real-time fluorescence quantitative PCR showed that compared with the blank control group and Negative Control si RNA transfection group,PINK1 si-2 group had the lowest PINK1 knockdown efficiency,so PINK1 si-2（hereinafter referred to as PINK1si RNA）was used for subsequent experiments（P<0.05）.5.PINK1 knockdown alleviates the morphological damage of SH-SY5Y cell processes caused by nonylphenol exposure:Crystal violet staining and optical microscopy of SH-SY5Y cells showed that compared with the blank Control group,the length and complexity of neural processes in the nonylphenol infected group were reduced,and compared with the Negative Control si RNA transfection+nonylphenol infected group,PINK1 si RNA transfection+nonylphenol infection group reduced the length and complexity of neurite.6.PINK1 knockdown alleviates overactivation of mitochondrial autophagy in SH-SY5Y cells induced by nonylphenol exposure:Laser confocal results showed that compared with the blank Control group,the fluorescence intensity of mitochondrial marker protein TOM20 in the nonylphenol infected group was weakened,and compared with the Negative Control si RNA transfection+nonylphenol infected group,The reduction of fluorescence intensity of mitochondrial marker protein TOM20 was alleviated in PINK1si RNA transfection+nonylphenol infection group.Immunofluorescence experiment results showed that,compared with the blank Control group,the fluorescence intensity of mitochondrial marker protein TIM23 in the nonylphenol infected group was weakened,and compared with the Negative Control si RNA transfection+nonylphenol infected group,The reduction of fluorescence intensity of mitochondrial marker protein TIM23 was alleviated in PINK1 si RNA transfection+nonylphenol infection group.Compared with the blank Control group,the fluorescence intensity of autophagy marker protein LC3 in the nonylphenol infected group was increased,and the increase of fluorescence intensity of autophagy marker protein LC3 in the PINK1 si RNA transfection+nonylphenol infected group was reduced compared with the Negative Control si RNA transfection+nonylphenol infected group.Compared with the blank Control group,the fluorescence intensity of the autophagy marker protein P62 in the nonylphenol infected group was decreased,and the fluorescence intensity of the autophagy marker protein P62 in the PINK1 si RNA transfected+nonylphenol infected group was alleviated compared with the Negative Control si RNA transfected+nonylphenol infected group.Western blot results showed that compared with the blank Control group,the expression level of mitochondrial marker protein TIM23 in the nonylphenol infected group was significantly decreased（P<0.05）,and compared with the Negative Control si RNA transfection+nonylphenol infected group,The expression level of mitochondrial marker protein TIM23 decreased after PINK1si RNA transfection and nonylphenol infection（P<0.05）.Compared with the blank Control group,the expression level of autophagy marker protein P62 in the nonylphenol infection group was significantly decreased（P<0.05）,and the expression level of autophagy marker protein P62 in the PINK1 si RNA transfection+nonylphenol infection group was alleviated compared with the Negative Control si RNA transfection+nonylphenol infection group（P<0.05）.Conclusion:1.Nonylphenol exposure led to dendrite damages in cerebellar Purkinje cells of rats.2.Nonylphenol exposure induced activation of PINK1/Parkin pathway and excessive mitophagy in cerebellar of rats.3.Mitophagy was involved in the dendrite damages of cerebellar Purkinje cells in rats exposed to nonylphenol.