| Objective: To observe the expression ofβ-1,4-GalT-I andβ-1,4-galactosylated carbohydrate chains in rat models of Central nervous systerm (CNS) inflammation and investigate their function in the process of inflammation in CNS.Methods: (1) The model of spinal cord inflammation was prepared in rats by the intraspinal injection of LPS. The expression ofβ-1,4-GalT-I mRNA andβ-1,4-galactosylated carbohydrate chains were assayed by real-time PCR and lectin-blot. The distribution and localization ofβ-1,4-GalT-I andβ-1,4-galactosylated carbohydrate chains in spinal cord were investigated by immunofluorescence staining. The location ofβ-1,4-GalT-I andβ-1,4-galactosylated carbohydrate chains were detected by methods of immunofluorescence double staining and lectin-fluorescent staining with RCA-I respectively. Double immunofluorescence staining and coimmunoprecipitation were used to study the interaction of E-selectin andβ-1,4-galactosylated carbohydrate chains. ELISA was used to detect the change of TNF-αprotein after intraspinal injection of LPS. RT-PCR was used to study the expression ofβ-1,4-GalT-I mRNA after LPS or TNF-αtreatment.(2) The models of PD were prepared by intranigral injection of LPS. Real-time PCR, Western-blot, Lectin-blott and double-label immunofluorescence were used to observe the expression and location ofβ-1,4-GalT-I mRNA andβ-1,4-galactosylated carbohydrate in SN.(3) An in vitro model relevant to PD—mixed neuron-glial culture were established using embryonic rat brain tissues from mesencephalon. Western-blot was used to detect the change ofβ-1,4-GalT-I protein expression after different exposure time. LPS orβ-1,4-GalT-I-Ab were used to treat mixed neuron-glial culture. Immunofluorescence and ELISA were used to investigate the microglial activation and phagocytosis in neuron-glial cultures.Results: (1) After intraspinal injection of LPS, the expression ofβ-1,4-GalT-I mRNA is time-dependent in spinal cord. The expression ofβ-1,4-GalT-I mRNA peaked at 6-8 h after LPS administration. LPS also induced expression ofβ-1,4-GalT-I protein andβ-1,4-galactosylated carbohydrate in the spinal cord to the higher lever. They expressed widespreadly and strongly both in the left grey matter and the dorsal column at 24 h after focal injection. Morphology analysis observed thatβ-1,4-GalT-I andβ-1,4-galactosylated carbohydrate were mostly expressed in actived microglia, neutrophils, and macrophages. Double immunofluorescence staining indicated that E-selectin co-expressed withβ-1,4-galactosylated carbohydrate. Coimmunoprecipitation further showed that E-selectin andβ-1,4-galactosylated carbohydrate interacted with each other in the damaged spinal cord. ELISA found TNF-αprotein content of the spinal cord increased with time after LPS injection. The co-localization ofβ-1,4-GalT-I and TNF-αwere also observed in animals at 24 h after surgery. LPS or TNF-αinducesβ-1,4-GalT-I mRNA expression in HAPI in a dose-dependent and time-dependent manner.(2) In normal rats,β-1,4-GalT-I mRNA was only weakly expressed,β-1,4-GalT-I mRNA expression reached plateau values at 8 h after intranigral injection of LPS, and then decreased gradually. Western blot analysis revealed thatβ-1,4-GalT-I protein was increased at 2 h after surgery, and remained elevated until 3 d after the surgery.β-1,4-galactosylated carbohydrate chains were also increased after LPS injection. Morphology analysis showed thatβ-1,4-GalT-I protein andβ-1,4-galactosylated carbohydrate chains were primarily expressed in actived microglia.(3) The expression ofβ-1,4-GalT-I protein was also detected in mixed neuron-glial culture by LPS treatment. Western-blotting revealed thatβ-1,4-GalT-I expression was significantly increased at 8 h, and remained elevated until 18 h after LPS treatment. Immunofluorescence staining showed thatβ-1,4-GalT-I-Ab attenuated LPS-increased microglial activation,release of TNF-αand phagocytosis.Conclusions: (1)β-1,4-GalT-I may play an important role in CNS inflammation induced by intraspianal injection of LPS and intranigral injection of LPS, especially participate in the phase of leukocyte recruitment during inflammation.(2) Up-regulation ofβ-1,4-GalT-I is attribute to a direct effect of TNF-αsignaling by the local injection of LPS.(3)β-1,4-GalT-I may play an important role in mediating microgliosis during CNS inflammation.
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