Background: The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes(PBLs) at the time of maternal recognition in cows. Blood samples were col...Background: The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes(PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination(AI). Based on the day of return of estrus, cows were divided into three groups, pregnant(n = 5), early embryonic mortality(EEM; n = 5) and late embryonic mortality(LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR.Results: The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d(P < 0.05), whereas no significant changes were observed both in EEM and LEM cows. Interferon-stimulated protein 15 k Da(ISG15), myxovirus-resistance gene(MX) 1 and MX2 mRNA expression in PBLs increased from 14 to 18 d which was significant on 18 d of pregnant cows as well as in LEM cows(P < 0.05), but no changes were observed in EEM cows. To determine whether the expression of CCL8 and CXCL10 in PBLs was regulated by pregnancy-related substances or not, expression level was assessed after exposure to interferon-τ(IFNT) and CCL16. Monocytes, granulocytes and lymphocytes were obtained using density-gradient centrifugation and flow cytometry. The addition of IFNT(100 ng/mL) and CCL16(100 ng/mL) to cultured PBLs increased the expression of CCL8 and CXCL10 mRNA(P < 0.05). The expression of ISG15, MX1 and MX2 mRNA in PBLs was also stimulated by IFNT and CCL16(P < 0.05).Conclusions: The expression of CCL8 and CXCL10 genes increased in PBLs during early pregnancy. Since IFNT stimulated CCL8 and CXCL10 expression in cultured PBLs, the increase of CCL8 and CXCL10 might be pregnancy-dependent events.The expression of both CCL8 and CXCL10 in PBLs was stimulated by CCL16 as wel as IFNT, suggesting a chemokine interaction that at least includes CCL8, CXCL10 and CCL16, and may play a role in regulating maternal recognition in cows.展开更多
OBJECTIVE Astrocytic gap junctions formed by connexin 43(Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders include chronic pain are associated with dysfun...OBJECTIVE Astrocytic gap junctions formed by connexin 43(Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders include chronic pain are associated with dysfunctional Cx43-gap junctions. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ) decreased both Cx43 expression and gap junction intercellular communication(GJIC)via a c-jun terminal kinase-dependent pathway. However,the exact mechanism that Cx43 does this in the context of spinal astrocytic dysfunction is unclear. The current study investigated the downstream signaling of Cx43-gap junction in rat primary cultured spinal astrocytes stimulated with cytokines.METHODS Wefocused on the glutamate transporters,examined the alteration of GLT-1 and glutamate-aspartate transporter(GLAST)expression and function in rat primary cultured spinal astrocytes stimulated with cytokines by real-time PCR and Western blotting. The function of GLT-1 was analyzed using the carbon 14. To elucidate the effect of Cx43 on glutamate transporters in spinal astrocytes,changes in glutamate transporters expression and function were quantified after Cx43 siRNA treatment.RESULTS The transcriptional and translational levels of Cx43 were reduced after 12 hr co-treatment with TNF-α(10 ng·mL^(-1)) or IFN-γ(5 ng·mL^(-1)). Furthermore,transcriptional and translational levels of GLT-1 and GLAST were also significantly reduced 24 and 48 h co-treatment with TNF-α or IFN-γ.Moreover,functional GLT-1 and GLAST uptake were inhibited by the mixture of TNF-α and IFN-γ. In addition,Both the decrease of GLT-1 expression and the reduction in functional GLT-1 uptake induced by the Cx43 si RNA,but both the expression and functional GLAST were no changes. CONCLUSION These results indicate that a Cx43 downregulation is induced under inflammatory condition that disrupts spinal astrocytic GLT-1 expression and function,leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state.展开更多
基金supported by Grants-in-Aid for the Research Program on Innovative Technologies for Animal Breeding,Reproduction,and Vaccine Development (REP1001) from the Ministry of Agriculture,Forestry and Fisheries of Japansupported by JSPS KAKENHI Grant Number 17 K08056
文摘Background: The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes(PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination(AI). Based on the day of return of estrus, cows were divided into three groups, pregnant(n = 5), early embryonic mortality(EEM; n = 5) and late embryonic mortality(LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR.Results: The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d(P < 0.05), whereas no significant changes were observed both in EEM and LEM cows. Interferon-stimulated protein 15 k Da(ISG15), myxovirus-resistance gene(MX) 1 and MX2 mRNA expression in PBLs increased from 14 to 18 d which was significant on 18 d of pregnant cows as well as in LEM cows(P < 0.05), but no changes were observed in EEM cows. To determine whether the expression of CCL8 and CXCL10 in PBLs was regulated by pregnancy-related substances or not, expression level was assessed after exposure to interferon-τ(IFNT) and CCL16. Monocytes, granulocytes and lymphocytes were obtained using density-gradient centrifugation and flow cytometry. The addition of IFNT(100 ng/mL) and CCL16(100 ng/mL) to cultured PBLs increased the expression of CCL8 and CXCL10 mRNA(P < 0.05). The expression of ISG15, MX1 and MX2 mRNA in PBLs was also stimulated by IFNT and CCL16(P < 0.05).Conclusions: The expression of CCL8 and CXCL10 genes increased in PBLs during early pregnancy. Since IFNT stimulated CCL8 and CXCL10 expression in cultured PBLs, the increase of CCL8 and CXCL10 might be pregnancy-dependent events.The expression of both CCL8 and CXCL10 in PBLs was stimulated by CCL16 as wel as IFNT, suggesting a chemokine interaction that at least includes CCL8, CXCL10 and CCL16, and may play a role in regulating maternal recognition in cows.
文摘OBJECTIVE Astrocytic gap junctions formed by connexin 43(Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders include chronic pain are associated with dysfunctional Cx43-gap junctions. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ) decreased both Cx43 expression and gap junction intercellular communication(GJIC)via a c-jun terminal kinase-dependent pathway. However,the exact mechanism that Cx43 does this in the context of spinal astrocytic dysfunction is unclear. The current study investigated the downstream signaling of Cx43-gap junction in rat primary cultured spinal astrocytes stimulated with cytokines.METHODS Wefocused on the glutamate transporters,examined the alteration of GLT-1 and glutamate-aspartate transporter(GLAST)expression and function in rat primary cultured spinal astrocytes stimulated with cytokines by real-time PCR and Western blotting. The function of GLT-1 was analyzed using the carbon 14. To elucidate the effect of Cx43 on glutamate transporters in spinal astrocytes,changes in glutamate transporters expression and function were quantified after Cx43 siRNA treatment.RESULTS The transcriptional and translational levels of Cx43 were reduced after 12 hr co-treatment with TNF-α(10 ng·mL^(-1)) or IFN-γ(5 ng·mL^(-1)). Furthermore,transcriptional and translational levels of GLT-1 and GLAST were also significantly reduced 24 and 48 h co-treatment with TNF-α or IFN-γ.Moreover,functional GLT-1 and GLAST uptake were inhibited by the mixture of TNF-α and IFN-γ. In addition,Both the decrease of GLT-1 expression and the reduction in functional GLT-1 uptake induced by the Cx43 si RNA,but both the expression and functional GLAST were no changes. CONCLUSION These results indicate that a Cx43 downregulation is induced under inflammatory condition that disrupts spinal astrocytic GLT-1 expression and function,leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state.