Inflammation plays an important role in nerve defects caused by intracerebral hemorrhage. Repairing brain damage by inhibiting the macrophage-inducible C-type lectin/spleen tyrosine kinase （Mincle/Syk） signaling pat...Inflammation plays an important role in nerve defects caused by intracerebral hemorrhage. Repairing brain damage by inhibiting the macrophage-inducible C-type lectin/spleen tyrosine kinase （Mincle/Syk） signaling pathway is a potential new target for treating cerebral hemorrhage. In this study, we aimed to determine whether acupuncture through Baihui （DU20） to Qubin （GBT） is an effective treatment for intracerebral hemorrhage through the Mincle/Syk signaling pathway. An intracerebral hemorrhage rat model was established by autol- ogous blood infusion into the caudate nucleus. Acupuncture through Baihui to Qubin was performed for 30 minutes, once every 12 hours, for a total of three times. Piceatannol （34.62 mg/kg）, a Syk inhibitor, was intraperitoneally injected as a control. Modified neurological severity score was used to assess neurological function. Brain water content was measured. Immunohistochemistry and western blot assay were used to detect immunoreactivity and protein expression levels of Minde, Syk, and CARD9. Real-time polymerase chain reaction was used to determine interleukin-1[~ mRNA levels. Hematoxylin-eosin staining was performed to observe histopathological changes. Our re- suits showed that acupuncture through Baihui to Qubin remarkably improved neurological function and brain water content, and inhibited immunoreactivity and expression of Mincle, Syk, CARDg, and interkeukin-1β Moreover, this effect was similar to piceatannol. These find- ings suggest that acupuncture through Baihui to Qubin can improve neurological impairment after cerebral hemorrhage by inhibiting the Mincle/Syk signaling pathway.展开更多
Background:TOSO,also named Fas inhibitory molecule 3(FAIM3),has recently been identified as an immunoglobulin M(IgM)Fc receptor(FcμR).Previous studies have shown that TOSO is specifically over-expressed in chronic ly...Background:TOSO,also named Fas inhibitory molecule 3(FAIM3),has recently been identified as an immunoglobulin M(IgM)Fc receptor(FcμR).Previous studies have shown that TOSO is specifically over-expressed in chronic lymphocytic leukemia(CLL).However,the functions of TOSO in CLL remain unknown.The B-cell receptor(BCR)signaling pathway has been reported to be constitutively activated in CLL.Here,we aimed to investigate the functions of TOSO in the BCR signaling pathway and the pathogenesis of CLL.Methods:We over-expressed TOSO in B-cell lymphoma cell lines(Granta-519 and Z138)by lentiviral transduction and knocked down TOSO by siRNA in primary CLL cells.The over-expression and knockdown of TOSO were confirmed at the RNA level by polymerase chain reaction and protein level by Western blotting.Co-immunoprecipitation with TOSO antibody followed by liquid chromatography coupled with tandem mass spectrometry(IP/LCMS)was used to identify TOSO interacting proteins.Western blotting was performed to detect the activation status of BCR signaling pathways as well as B-cell lymphoma 2(BCL-2).Flow cytometry was used to examine the apoptosis of TOSO-over-expressing B lymphoma cell lines and TOSO-down-regulated CLL cells via the staining of Annexin V and 7-AAD.One-way analyses of variance were used for intergroup comparisons,while independent samples t tests were used for two-sample comparisons.Results:From IP/LCMS,we identified spleen tyrosine kinase(SYK)as a crucial candidate of TOSO-interacting protein and confirmed it by co-immunoprecipitation.After stimulation with anti-IgM,TOSO over-expression increased the phosphorylation of SYK,and subsequently activated the BCR signaling pathway,which could be reversed by a SYK inhibitor.TOSO knockdown in primary CLL cells resulted in reduced SYK phosphorylation as well as attenuated BCR signaling pathway.The apoptosis rates of the Granta-519 and Z138 cells expressing TOSO were(8.46±2.90)%and(4.20±1.21)%,respectively,significantly lower than the rates of the control groups,which were(25.20±4.60)%and(19.72±1.10)%,respectively(P<0.05 for both).The apoptosis rate was reduced after knocking down TOSO in the primary CLL cells.In addition,we also found that TOSO down-regulation in primary cells from CLL patients led to decreased expression of BCL-2 as well as lower apoptosis,and vice versa in the cell line.Conclusions:TOSO might be involved in the pathogenesis of CLL by interacting with SYK,enhancing the BCR signaling pathway,and inducing apoptosis resistance.展开更多
基金supported by the National Natural Science Foundation of China,No.81473764,81273824the Key Project of Natural Science Foundation of Heilongjiang Province of China,No.ZD201204the Doctoral Fund Program of Ministry of Education of China,No.20102327110003
文摘Inflammation plays an important role in nerve defects caused by intracerebral hemorrhage. Repairing brain damage by inhibiting the macrophage-inducible C-type lectin/spleen tyrosine kinase （Mincle/Syk） signaling pathway is a potential new target for treating cerebral hemorrhage. In this study, we aimed to determine whether acupuncture through Baihui （DU20） to Qubin （GBT） is an effective treatment for intracerebral hemorrhage through the Mincle/Syk signaling pathway. An intracerebral hemorrhage rat model was established by autol- ogous blood infusion into the caudate nucleus. Acupuncture through Baihui to Qubin was performed for 30 minutes, once every 12 hours, for a total of three times. Piceatannol （34.62 mg/kg）, a Syk inhibitor, was intraperitoneally injected as a control. Modified neurological severity score was used to assess neurological function. Brain water content was measured. Immunohistochemistry and western blot assay were used to detect immunoreactivity and protein expression levels of Minde, Syk, and CARD9. Real-time polymerase chain reaction was used to determine interleukin-1[~ mRNA levels. Hematoxylin-eosin staining was performed to observe histopathological changes. Our re- suits showed that acupuncture through Baihui to Qubin remarkably improved neurological function and brain water content, and inhibited immunoreactivity and expression of Mincle, Syk, CARDg, and interkeukin-1β Moreover, this effect was similar to piceatannol. These find- ings suggest that acupuncture through Baihui to Qubin can improve neurological impairment after cerebral hemorrhage by inhibiting the Mincle/Syk signaling pathway.
基金This work was supported by grants from the National Nature Science Foundation of China(Nos.81200395 and 81970187)Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences grants(No.2017-I2M-3-018)Fundamental Application and Advanced and Technology Research Program of Tianjin(No.15JCYBJC25100)。
文摘Background:TOSO,also named Fas inhibitory molecule 3(FAIM3),has recently been identified as an immunoglobulin M(IgM)Fc receptor(FcμR).Previous studies have shown that TOSO is specifically over-expressed in chronic lymphocytic leukemia(CLL).However,the functions of TOSO in CLL remain unknown.The B-cell receptor(BCR)signaling pathway has been reported to be constitutively activated in CLL.Here,we aimed to investigate the functions of TOSO in the BCR signaling pathway and the pathogenesis of CLL.Methods:We over-expressed TOSO in B-cell lymphoma cell lines(Granta-519 and Z138)by lentiviral transduction and knocked down TOSO by siRNA in primary CLL cells.The over-expression and knockdown of TOSO were confirmed at the RNA level by polymerase chain reaction and protein level by Western blotting.Co-immunoprecipitation with TOSO antibody followed by liquid chromatography coupled with tandem mass spectrometry(IP/LCMS)was used to identify TOSO interacting proteins.Western blotting was performed to detect the activation status of BCR signaling pathways as well as B-cell lymphoma 2(BCL-2).Flow cytometry was used to examine the apoptosis of TOSO-over-expressing B lymphoma cell lines and TOSO-down-regulated CLL cells via the staining of Annexin V and 7-AAD.One-way analyses of variance were used for intergroup comparisons,while independent samples t tests were used for two-sample comparisons.Results:From IP/LCMS,we identified spleen tyrosine kinase(SYK)as a crucial candidate of TOSO-interacting protein and confirmed it by co-immunoprecipitation.After stimulation with anti-IgM,TOSO over-expression increased the phosphorylation of SYK,and subsequently activated the BCR signaling pathway,which could be reversed by a SYK inhibitor.TOSO knockdown in primary CLL cells resulted in reduced SYK phosphorylation as well as attenuated BCR signaling pathway.The apoptosis rates of the Granta-519 and Z138 cells expressing TOSO were(8.46±2.90)%and(4.20±1.21)%,respectively,significantly lower than the rates of the control groups,which were(25.20±4.60)%and(19.72±1.10)%,respectively(P<0.05 for both).The apoptosis rate was reduced after knocking down TOSO in the primary CLL cells.In addition,we also found that TOSO down-regulation in primary cells from CLL patients led to decreased expression of BCL-2 as well as lower apoptosis,and vice versa in the cell line.Conclusions:TOSO might be involved in the pathogenesis of CLL by interacting with SYK,enhancing the BCR signaling pathway,and inducing apoptosis resistance.
