AIM To investigate the effects of herb-partitioned moxibustion(HPM) on phosphorylation of mitogen-activated extracellular signal-regulated kinase(MEK)1, extracellular signal-regulated kinase(ERK)1/2 and c AMP response...AIM To investigate the effects of herb-partitioned moxibustion(HPM) on phosphorylation of mitogen-activated extracellular signal-regulated kinase(MEK)1, extracellular signal-regulated kinase(ERK)1/2 and c AMP response element binding protein(CREB) in spinal cord of rats with chronic inflammatory visceral pain(CIVP), and to explore the central mechanism of HPM in treating CIVP.METHODS Male Sprague-Dawley rats were randomized into normal, model, HPM, sham-HPM, MEK-inhibitor and dimethyl sulfoxide(DMSO) groups. The CIVP model was established using an enema mixture of trinitrobenzene sulfonic acid and ethanol. HPM was applied at bilateral Tianshu(ST25) and Qihai(CV6) acupoints in the HPM group, while in the sham-HPM group, moxa cones and herb cakes were only placed on the same points but not ignited. The MEK-inhibitor and DMSO groups received L5-L6 intrathecal injection of U0126 and 30% DMSO, respectively. Abdominal withdrawal reflex(AWR), mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) were applied for the assessment of pain behavior. The colonic tissue was observed under an optical microscope after hematoxylin-eosin staining. Expression of phosphor(p)MEK1, p ERK1/2 and p CREB in rat spinal cord was detected using Western blotting. The levels of MEK, ERK and CREB m RNA in rat spinal cord were detected using real-time polymerase chain reaction. RESULTS Compared with the normal group, the AWR scores were increased significantly(P < 0.01) and the MWT and TWL scores were decreased significantly(P < 0.05) in the model, sham-HPM and DMSO groups. Compared with the model group, the AWR scores were decreased significantly(P < 0.01) and the MWT and TWL scores were increased significantly in the HPM and MEK-inhibitor groups(P < 0.05). Compared with the sham-HPM and DMSO groups, the AWR scores were decreased significantly(P < 0.01) and the MWT and TWL scores were increased significantly(P < 0.05) in the HPM and MEK-inhibitor groups. Compared with the normal group, the expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were increased significantly in the model, sham-HPM and DMSO groups(P < 0.01 or < 0.05). Compared with the model group, the expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups(P < 0.01 or < 0.05). Compared with the sham-HPM and DMSO groups, expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups(P < 0.01 or < 0.05). CONCLUSION HPM down-regulates protein phosphorylation of MEK1, ERK1/2 and CREB, and m RNA expression of MEK, ERK and CREB, inhibiting activation of the MEK/ERK/CREB signaling pathway in the spinal cord of CIVP rats, which is possibly a critical central mechanism of the analgesic effect of HPM.展开更多
This paper aims to observe the changes of the inflammatory cytokines in microglial BV2 cells stimulated by apelin, and inves- tigate the mechanism of inflammatory cytokines secretion after apelin stimulation. Immunofl...This paper aims to observe the changes of the inflammatory cytokines in microglial BV2 cells stimulated by apelin, and inves- tigate the mechanism of inflammatory cytokines secretion after apelin stimulation. Immunofluorescence and quantitative re- al-time PCR were performed to observe expression of TNF-α, IL-Iβ, IL-10, MIP-let, and MCP-1 in BV2 cells. Western blot was used to investigate the expression of phosphorylation PI-3K/Akt and phosphorylation Erk signaling pathways in BV2 cells after stimulation by apelin. Furthermore, PI-3K/Akt inhibitor （LY294402） and Erk inhibitor （U0126） were used as antagonists to detect the secretion mechanisms of cytokines in BV2 cells stimulated by apelin. Exogenous recombinant apelin activated the expression of TNF-α, IL-1β, MCP-1 and MIP-1α in BV2 cells by the detection of fluorescence expression and mRNA. Apelin also unregulated the protein expression of p-PI-3K/Akt and p-Erk in BV2 cells induced by apelin. LY294002 and U0126 in- hibited activation of p-PI-3K/Akt and p-Erk expression by Western blot and attenuated the expression of inflammation factors in BV2 cells by fluorescence staining. This study demonstrates that apelin is a potential activator of inflammation factors through the PI3K/Akt and Erk signaling pathway and is potential therapeutically relevant to inflammatory responses of micro- glia cells.展开更多
基金Supported by National Natural Science Foundation of China,No.81273843 and No.81674073National Key Basic Research Program of China(973 Program)+1 种基金No.2015CB554501Project of Shanghai Municipal Commission of Health and Family Planning,No.20144Y0153 and No.2017BR047
文摘AIM To investigate the effects of herb-partitioned moxibustion(HPM) on phosphorylation of mitogen-activated extracellular signal-regulated kinase(MEK)1, extracellular signal-regulated kinase(ERK)1/2 and c AMP response element binding protein(CREB) in spinal cord of rats with chronic inflammatory visceral pain(CIVP), and to explore the central mechanism of HPM in treating CIVP.METHODS Male Sprague-Dawley rats were randomized into normal, model, HPM, sham-HPM, MEK-inhibitor and dimethyl sulfoxide(DMSO) groups. The CIVP model was established using an enema mixture of trinitrobenzene sulfonic acid and ethanol. HPM was applied at bilateral Tianshu(ST25) and Qihai(CV6) acupoints in the HPM group, while in the sham-HPM group, moxa cones and herb cakes were only placed on the same points but not ignited. The MEK-inhibitor and DMSO groups received L5-L6 intrathecal injection of U0126 and 30% DMSO, respectively. Abdominal withdrawal reflex(AWR), mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) were applied for the assessment of pain behavior. The colonic tissue was observed under an optical microscope after hematoxylin-eosin staining. Expression of phosphor(p)MEK1, p ERK1/2 and p CREB in rat spinal cord was detected using Western blotting. The levels of MEK, ERK and CREB m RNA in rat spinal cord were detected using real-time polymerase chain reaction. RESULTS Compared with the normal group, the AWR scores were increased significantly(P < 0.01) and the MWT and TWL scores were decreased significantly(P < 0.05) in the model, sham-HPM and DMSO groups. Compared with the model group, the AWR scores were decreased significantly(P < 0.01) and the MWT and TWL scores were increased significantly in the HPM and MEK-inhibitor groups(P < 0.05). Compared with the sham-HPM and DMSO groups, the AWR scores were decreased significantly(P < 0.01) and the MWT and TWL scores were increased significantly(P < 0.05) in the HPM and MEK-inhibitor groups. Compared with the normal group, the expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were increased significantly in the model, sham-HPM and DMSO groups(P < 0.01 or < 0.05). Compared with the model group, the expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups(P < 0.01 or < 0.05). Compared with the sham-HPM and DMSO groups, expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups(P < 0.01 or < 0.05). CONCLUSION HPM down-regulates protein phosphorylation of MEK1, ERK1/2 and CREB, and m RNA expression of MEK, ERK and CREB, inhibiting activation of the MEK/ERK/CREB signaling pathway in the spinal cord of CIVP rats, which is possibly a critical central mechanism of the analgesic effect of HPM.
基金supported by European Foundation for the Study of Diabetes/Chinese Diabetes Society/Lilly grant(90561 and 94410)
文摘This paper aims to observe the changes of the inflammatory cytokines in microglial BV2 cells stimulated by apelin, and inves- tigate the mechanism of inflammatory cytokines secretion after apelin stimulation. Immunofluorescence and quantitative re- al-time PCR were performed to observe expression of TNF-α, IL-Iβ, IL-10, MIP-let, and MCP-1 in BV2 cells. Western blot was used to investigate the expression of phosphorylation PI-3K/Akt and phosphorylation Erk signaling pathways in BV2 cells after stimulation by apelin. Furthermore, PI-3K/Akt inhibitor （LY294402） and Erk inhibitor （U0126） were used as antagonists to detect the secretion mechanisms of cytokines in BV2 cells stimulated by apelin. Exogenous recombinant apelin activated the expression of TNF-α, IL-1β, MCP-1 and MIP-1α in BV2 cells by the detection of fluorescence expression and mRNA. Apelin also unregulated the protein expression of p-PI-3K/Akt and p-Erk in BV2 cells induced by apelin. LY294002 and U0126 in- hibited activation of p-PI-3K/Akt and p-Erk expression by Western blot and attenuated the expression of inflammation factors in BV2 cells by fluorescence staining. This study demonstrates that apelin is a potential activator of inflammation factors through the PI3K/Akt and Erk signaling pathway and is potential therapeutically relevant to inflammatory responses of micro- glia cells.
