Full
Outline
To investigate the mechanism by which suppressor of cytokine signaling 3 (SOCS3) regulates microglial inflammation through nuclear factor-kappaB (NF-κB), providing novel mechanistic insights into microglial involvement in Parkinson’s disease (PD) pathogenesis.
① Ten male C57BL/6 mice (12 weeks old, weighing 20~25 g) were subjected to intraperitoneal injection of 15 mg/kg MPTP to establish a PD model. Rotarod test was used to assess motor function. Western blotting was employed to detect the protein expression of tyrosine hydroxylase (TH) and ionized calcium-binding adapter molecule 1 (IBA-1) in the substantia nigra. RT-qPCR was utilized to measure the mRNA level of SOCS3 in the substantia nigra. Immunohistochemistry was performed to assess NF-κB p65 subunit expression. The expression of SOCS3, NF-κB and p-NF-κB was measured with Western blotting. ② Microglial cell line BV2 was stimulated with 1 000 ng/mL lipopolysaccharide (LPS) for 6 h to establish an inflammatory model. Subsequently, SOCS3 was knocked down. NF-κB inhibitor BAY 11-7082 was used to treat the cells. RT-qPCR and Western blotting were used to measure the expression of SOCS3 at mRNA and protein levels. Western blotting was also applied to detect the expression of NF-κB and p-NF-κB, and ELISA was conducted to measure TNF-α and IL-1β levels in the culture supernatant. Immunofluorescence assay was carried out to localize NF-κB (nuclear vs cytoplasmic). ③ A co-culture system of BV2 microglia and N2a neuroblastoma cells was established to investigate the regulatory effects of microglia on neuronal cells. MTT assay and TUNEL staining were used respectively to determine cell viability and apoptosis of N2a cells.
① Compared to the control mice, the PD mouse model exhibited reduced rotarod fall latency, down-regulation in TH and SOCS3(P<0.01), up-regulation in IBA-1 and increased p-NF-κB/NF-κB ratio (P<0.01). ② In BV2 cells, LPS stimulation increased TNF-α, IL-1β, and p-NF-κB/NF-κB ratio(P<0.01), while down-regulated SOCS3 expression(P<0.01). SOCS3 knockdown in LPS-stimulated BV2 cells further increased the p-NF-κB/NF-κB ratio (P<0.01), increased nuclear localization of NF-κB, and elevated TNF-α and IL-1β levels (P<0.01). BAY 11-7082 treatment in these SOCS3-knockdown, LPS-stimulated cells resulted in reduced p-NF-κB/NF-κB ratio, TNF-α, and IL-1β (P<0.01), and decreased NF-κB nuclear distribution. ③ LPS-stimulated BV2 cells reduced cell viability and increased cell apoptosis in N2a cells(P<0.01). SOCS3 knockdown in BV2 cells exacerbated the reduction in N2a cell viability (P<0.01) and the increase in cell apoptosis in N2a cells (P<0.01). BAY 11-7082 treatment of these SOCS3-knockdown BV2 microglia attenuated the reduction in N2a cell viability and decreased apoptosis in N2a cells (P<0.01).
SOCS3 inhibits microglia inflammatory response through down-regulation of NF-kB activity, and in turn attenuates neuronal cell death and ameliorates PD nerve injury.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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