Abstract：
BACKGROUND: Oxidative stress and neuroinflammation are proven to play critical roles in the pathogenesis of Parkinson\'s disease (PD). As reported, patients with PD have lower level of STAT4 compared with healthy subjects. However, the biological functions and mechanisms of STAT4 in PD pathogenesis remain uncertain. This study aimed to investigate the roles and related mechanisms of STAT4 in PD development.
METHODS: The intraperitoneal injection of MPTP (20 mg/kg) dissolved in physiological saline was performed to mimic PD-like conditions in vivo. MPP + solution was prepared for cell model of PD. Cell viability was measured by CCK-8. Griess reaction was conducted to measure NO concentrations. The mRNA and protein levels were evaluated by RT-qPCR and western blotting. ROS generation was assessed by DCFH-DA. The levels of inflammatory cytokines were measured by ELISA. Cell apoptosis was examined by flow cytometry and western blotting. Moreover, the SH-SY5Y cells were treated with conditioned medium from LPS-stimulated microglia and subjected to CCK-8 assays and ELISA. Mechanistically, CHIP assays and luciferase reporter assays were performed to verify the binding relationship between KISS1 and STAT4. For in vivo analysis, the histological changes of midbrain tissues of mice were determined by hematoxylin and eosin staining. The expression of tyrosine hydroxylase (TH) was detected by immunohistochemistry staining. Iba-1 positive microglial cells in the striatum were assessed by immunofluorescence staining.
RESULTS: For in vitro analysis, STAT4 level was downregulated after MPP+ treatment, and STAT4 upregulation inhibited the oxidative damage, inflammation and apoptosis in SH-SY5Y cells. STAT4 bound at +215-228 region of KISS1, and KISS1 upregulation counteracted the protection of STAT4 upregulation against cell damage. Moreover, STAT4 upregulation inhibited cell viability loss and inflammation induced by conditioned medium from LPS-treated microglia, whereas KISS1 upregulation had the opposite effect. For in vivo analysis, the protective effects of STAT4 upregulation against inflammatory response, oxidative stress, dopaminergic neuronal loss and microglia activation were attenuated by KISS1 upregulation. Moreover, the inactivation of MAPK pathway caused by STAT4 upregulation was reversed by KISS1 upregulation, and MAPK inhibition attenuated the MPP+-induced inflammation, oxidative stress and apoptosis in SH-SY5Y cells.
CONCLUSIONS: STAT4 inhibits KISS1 to attenuate the oxidative damage, inflammation and neuronal apoptosis in PD by inactivating the MAPK pathway.
METHODS: The intraperitoneal injection of MPTP (20 mg/kg) dissolved in physiological saline was performed to mimic PD-like conditions in vivo. MPP + solution was prepared for cell model of PD. Cell viability was measured by CCK-8. Griess reaction was conducted to measure NO concentrations. The mRNA and protein levels were evaluated by RT-qPCR and western blotting. ROS generation was assessed by DCFH-DA. The levels of inflammatory cytokines were measured by ELISA. Cell apoptosis was examined by flow cytometry and western blotting. Moreover, the SH-SY5Y cells were treated with conditioned medium from LPS-stimulated microglia and subjected to CCK-8 assays and ELISA. Mechanistically, CHIP assays and luciferase reporter assays were performed to verify the binding relationship between KISS1 and STAT4. For in vivo analysis, the histological changes of midbrain tissues of mice were determined by hematoxylin and eosin staining. The expression of tyrosine hydroxylase (TH) was detected by immunohistochemistry staining. Iba-1 positive microglial cells in the striatum were assessed by immunofluorescence staining.
RESULTS: For in vitro analysis, STAT4 level was downregulated after MPP+ treatment, and STAT4 upregulation inhibited the oxidative damage, inflammation and apoptosis in SH-SY5Y cells. STAT4 bound at +215-228 region of KISS1, and KISS1 upregulation counteracted the protection of STAT4 upregulation against cell damage. Moreover, STAT4 upregulation inhibited cell viability loss and inflammation induced by conditioned medium from LPS-treated microglia, whereas KISS1 upregulation had the opposite effect. For in vivo analysis, the protective effects of STAT4 upregulation against inflammatory response, oxidative stress, dopaminergic neuronal loss and microglia activation were attenuated by KISS1 upregulation. Moreover, the inactivation of MAPK pathway caused by STAT4 upregulation was reversed by KISS1 upregulation, and MAPK inhibition attenuated the MPP+-induced inflammation, oxidative stress and apoptosis in SH-SY5Y cells.
CONCLUSIONS: STAT4 inhibits KISS1 to attenuate the oxidative damage, inflammation and neuronal apoptosis in PD by inactivating the MAPK pathway.
摘要：
背景：氧化应激和神经炎症已被证明在帕金森病（PD）的发病机制中起关键作用。据报道，与健康受试者相比，PD患者的STAT4水平较低。然而,STAT4在PD发病中的生物学功能和机制尚不明确。本研究旨在探讨STAT4在PD发生发展中的作用及相关机制。
方法：腹膜内注射溶解在生理盐水中的MPTP（20mg/kg）以模拟体内PD样条件。制备用于PD细胞模型的MPP+溶液。通过CCK-8测量细胞活力。进行Griess反应以测量NO浓度。通过RT-qPCR和蛋白质印迹评估mRNA和蛋白质水平。通过DCFH-DA评估ROS生成。通过ELISA测量炎性细胞因子的水平。通过流式细胞术和蛋白质印迹法检查细胞凋亡。此外，用来自LPS刺激的小胶质细胞的条件培养基处理SH-SY5Y细胞,并进行CCK-8测定和ELISA。机械上,进行CHIP测定和荧光素酶报告基因测定以验证KISS1和STAT4之间的结合关系。对于体内分析，用苏木精和伊红染色测定小鼠中脑组织的组织学变化。免疫组化染色检测酪氨酸羟化酶(TH)的表达。通过免疫荧光染色评估纹状体中的Iba-1阳性小胶质细胞。
结果：对于体外分析，MPP+治疗后STAT4水平下调,STAT4上调抑制氧化损伤，SH-SY5Y细胞的炎症和凋亡。STAT4在KISS1的+215-228区域结合,并且KISS1上调抵消了STAT4上调对细胞损伤的保护。此外，STAT4上调抑制了LPS处理的小胶质细胞条件培养基诱导的细胞活力丧失和炎症，而KISS1上调则有相反的作用。对于体内分析，STAT4上调对炎症反应的保护作用，氧化应激，多巴胺能神经元丢失和小胶质细胞激活通过KISS1上调而减弱。此外，STAT4上调引起的MAPK通路失活被KISS1上调逆转,和MAPK抑制减弱MPP+诱导的炎症，SH-SY5Y细胞的氧化应激和凋亡。
结论：STAT4抑制KISS1减轻氧化损伤，通过灭活MAPK通路在PD中的炎症和神经元凋亡。
方法：腹膜内注射溶解在生理盐水中的MPTP（20mg/kg）以模拟体内PD样条件。制备用于PD细胞模型的MPP+溶液。通过CCK-8测量细胞活力。进行Griess反应以测量NO浓度。通过RT-qPCR和蛋白质印迹评估mRNA和蛋白质水平。通过DCFH-DA评估ROS生成。通过ELISA测量炎性细胞因子的水平。通过流式细胞术和蛋白质印迹法检查细胞凋亡。此外，用来自LPS刺激的小胶质细胞的条件培养基处理SH-SY5Y细胞,并进行CCK-8测定和ELISA。机械上,进行CHIP测定和荧光素酶报告基因测定以验证KISS1和STAT4之间的结合关系。对于体内分析，用苏木精和伊红染色测定小鼠中脑组织的组织学变化。免疫组化染色检测酪氨酸羟化酶(TH)的表达。通过免疫荧光染色评估纹状体中的Iba-1阳性小胶质细胞。
结果：对于体外分析，MPP+治疗后STAT4水平下调,STAT4上调抑制氧化损伤，SH-SY5Y细胞的炎症和凋亡。STAT4在KISS1的+215-228区域结合,并且KISS1上调抵消了STAT4上调对细胞损伤的保护。此外，STAT4上调抑制了LPS处理的小胶质细胞条件培养基诱导的细胞活力丧失和炎症，而KISS1上调则有相反的作用。对于体内分析，STAT4上调对炎症反应的保护作用，氧化应激，多巴胺能神经元丢失和小胶质细胞激活通过KISS1上调而减弱。此外，STAT4上调引起的MAPK通路失活被KISS1上调逆转,和MAPK抑制减弱MPP+诱导的炎症，SH-SY5Y细胞的氧化应激和凋亡。
结论：STAT4抑制KISS1减轻氧化损伤，通过灭活MAPK通路在PD中的炎症和神经元凋亡。
