| Cerebral ischemia is the most common type of cerebrovascular disease, which is the first leading cause of death and the most frequent cause of permanent disability in adults. Ischemic stroke is one of the main diseases endangering people’s health in our state, which has a high incidence, mortality, morbidity, high recurrence rate. Studies showed that cerebral ischemia triggered massive physiopathologic processes including energy metabolism, inflammatory responses, oxidative stress, free radical damage, excitatory amion acids toxicity, etc., which directly/indirectly results in neuronal apoptosis/death, eventually leading to neurological deficit.Among those inflammatory responses is one of the most important mechanisms. In ischemic brain injury, molecules released from injured tissue, blood vessels, and necrotic cells activate Toll-like receptors(TLRs) and induce production of proinflammatory cytokines, such as interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α). These cytokines cause postischemic inflammation in the brain, subsequently leading to exacerbated primary brain damage.The expression of inflammation-related genes can be regulated not only by transcription factors at the transcriptional level but also by micro RNAs(mi RNAs, mi Rs) at the posttranscriptional level. Emerging evidence suggest that mi RNAs are involved in regulating several aspects of inflammation and the response to cerebral ischemia. Among various mi RNAs, mi R-155 is an inflammation-related mi RNA, its expression is upregulated in cancer, cardiovascular disease, inflammation, autoimmune diseases and transplant rejection. Its expression is induced in inflammatory macrophages, it enhances proinflammatory cytokine expression in macrophages by regulating the NF-κB signaling pathway. In addition, mi R-155 can exert both pro- and anti-inflammatory effects by targeting different mediators of inflammatory signaling, such as SHIP1, SOCS1, SMAD2, and TAB2. Given the importance of mi R-155 in the development of postischemic brain inflammation, understanding the role of mi R-155 and mechanism of mi R-155 actions in ischemic cerebral injury will provide novel insights into ischemic stroke therapy.In recent years, based on the research of the neuroprotective agent, some natural or synthetic materials have been gradually discovered and confirmed to be protective against cerebral ischemia injury. Acetylbritannilactone(ABL) is a new anti-inflammatory compound isolated from Inula britannica L., a traditional Chinese medicinal herb. Several of the previous studies demonstrate that ABL inhibits the expression of inflammation-associated genes, such as the NOS and COX-2 genes, by reducing IκB-α phosphorylation and degradation, inhibiting NF-κB activation, and blocking the binding of active NF-κB to the target gene promoters in RAW 264.7 macrophages and vascular smooth muscle cells(VSMCs). In addition, ABL can also suppress PDGF-induced DNA synthesis and cell proliferation, subsequently leading to apoptosis in proliferative VSMCs via the induction of a higher ratio of Bax/Bcl-2, activation of caspase-9/-3, and the cleavage of the endogenous substrate Poly(ADP-ribose) polymerase.Although ABL exhibits potent anti-inflammatory and pro-apoptotic effects in macrophages and VSMCs, the effects of ABL on cerebral ischemia have not been evaluated. Simultaneously, it remains also unclear whether anti-inflammation effects of ABL on cerebral ischemia are mediated by mi R-155. The study was divided into three parts listed below. PartⅠABL exerts the protective effect on ischemic cerebral tissues by inhibiting the inflammatory responsesObjective: To observe the effects of ABL on inflammatory responses induced by focal cerebral ischemia in mice.Methods: Male, healthy CD1 mice were subjected to permanent middle cerebral artery occlusion(p MCAO). The mice were randomly divided into 5 groups: sham group, MCAO group, ABL-low dose(ABL-L) group, ABL-middle dose(ABL-M) group, ABL-high dose(ABL-H) group. Sham group: with the same operation to MCAO group except not inserting nylon line. ABL treatment groups: ABL was injected intraperitoneally 30 minutes prior to MCAO operation. ABL concentration was 10mg/kg in ABL-L group, 20mg/kg in ABL-M group and 30mg/kg in ABL-H goup.BV2 cells were divided into con group and oxygen-glucose deprivation(OGD) group, and ABL treatment group was pretreated with ABL(100 μmol/L) for 24 hours and then stimulated with different treatments.Mice were anesthetized and killed at 24 h after stroke, and neurological function, brain edema and infarct volume were measured. Immunohistochemistry, western blotting and q RT-PCR were used to examine the expression of TNF-α, IL-1β,NF-κB, TLR-4, I-κB, My D88 and SOCS1.Results:1 Neurological function scores of MCAO group were higher than that of sham group(P<0.05). Neurological function scores were significant reduced in ABL-M and ABL-H groups compared with MCAO group(P< 0.05). There was no significant difference between ABL-L and MCAO group(P > 0.05).2 Percentage of brain water content of MCAO group was higher than that of sham group(P < 0.05), and percentage of brain water content of ABL-H group and ABL-M group decreased compared with that of MCAO group. There was no significant difference between ABL-L group and MCAO group(P > 0.05).3 After TTC staining, the normal tissue was stained dark red while the infarct area pale gray color. ABL-treated mice showed smaller infarct volumes compared with MCAO group.4 ABL decreases inflammatory responses induced by focal cerebral ischemia: immunohistochemical staining results showed that the positive cells of TNF-α and IL-1β were significantly increased in MCAO group, and ABL treatment dramatically decreased the number of the positive cells. Western blotting results showed a significant increase in TNF-α and IL-1β in MCAO group(P < 0.05), ABL treatment markedly suppressed upregulation of TNF-α and IL-1β, especially in ABL-H group. q RT-PCR results showed that the m RNA expression of TNF-α and IL-1β was significantly increased in MCAO group, and ABL also significantly inhibited the m RNA expression. ELISA showed that the protein concentrations of TNF-α and IL-1β in brain tissues 24 hours after MCAO were markedly increased(P < 0.05). In BV2 cells, the levels of TNF-α and IL-1β were increased after OGD, 100 μmol/L of ABL significantly reduced the TNF-α and IL-1β levels in BV2 cells under OGD(P < 0.05).5 ABL decreases inflammatory responses via inhibiting the TLR4/NF-κB signaling cascades. Immunohistochemical staining results showed that the positive cells of TLR4 and NF-κB were significantly increased in MCAO group, and ABL treatment dramatically decreased the number of positive cells. Western blotting results showed that the expression levels of TLR4 and NF-κB were obviously increased in MCAO group(P < 0.05), and MCAO provoked a significant decrease in the expression of I-κB and TAB2. Compared to the MCAO group, TLR4 and NF-κB expression was significantly decreased in the three ABL-treated groups(P < 0.05), and the inhibitory effect of ABL-H on TLR4 and NF-κB expression was more significant than that of ABL-L. In contrast, ABL dose-dependently increased the expression of I-κB, My D88, and SOCS1(P < 0.05). A similar result was observed in OGD-treated BV2 cells, showing that ABL significantly reduced the NF-κB and TLR4 levels, but increased the levels of I-κB, My D88, and SOCS1(P < 0.05).Conclusions: ABL treatment effectively ameliorates neurological defect, brain edema and brain infarct volume. ABL dose-dependently inhibits TNF-α and IL-1β expression in ischemic cerebral tissue of MCAO mice and OGD-treated BV2 cells. ABL decreases inflammatory responses by suppressing the expression of the proinflammatory signaling molecules TLR4 and NF-κB. PartⅡThe expression of mi R-155 in the ischemic cerebral tissue and theeffects of ABL on mi R-155 expressionObjective: To examine whether mi R-155 is required for the inflammatory responses in the ischemic cerebral tissue, and whether ABL exerts its anti-inflammation action by regulating mi R-155 expression.Methods: Experiment 1: Male, healthy CD1 mice were subjected to permanent middle cerebral artery occlusion(p MCAO). ABL was injected intraperitoneally 30 minutes prior to MCAO operation. The mice were randomly divided into 5 groups: sham group, MCAO group, ABL-low dose(ABL-L) group, ABL-middle dose(ABL-M) group, ABL-high dose(ABL-H) group. BV2 cells were divided into con group and oxygen-glucose deprivation(OGD) group, and ABL treatment group was pretreated by ABL(100 μmol/L) for 24 hours and then stimulated with different treatments. Mice were anesthetized and killed at 24 h after stroke. q RT-PCR and reporter gene assays were used to examine the expression of mi R-155 in ischemic cerebral tissue and BV2 cells.Experiment 2: Male, 8-week-old mi R-155 KO and wild-type(WT) mice were subjected to permanent middle cerebral artery occlusion(p MCAO). p Ad-mi R-155(p Ad as a control) was introduced into WT mice through LV injection to overexpress mi R-155. ABL(20 mg/kg) was injected intraperitoneally 30 minutes prior to MCAO operation. BV2 microglial cells were used in vitro study. Adenovirus infection experiments were performed in BV2 cells to overexpress mi R-155. si RNA transfection experiments were performed in BV2 cells to knock down mi R-155. BV2 cells were treated with or without ABL(100 μmol/L), and then exposed to OGD for 4 hours. Mice were anesthetized and killed at 24 h after stroke. Neurological function and infarct volume were measured. Western blotting and q RT-PCR were used to examine the expression of TNF-α and IL-1β.Results:1 mi R-155 expression in ischemic cerebral tissue and BV2 microglial cells: q RT-PCR results showed that mi R-155 was markedly upregulated in ischemic cerebral tissues after 24 hours of MCAO(P<0.05). ABL treatment dose-dependently suppressed the expression of mi R-155 induced by MCAO(P<0.05). In OGD-treated BV2 cells, mi R-155 upregulation induced by OGD was abolished by treating BV2 cells with 100 μmol/L of ABL(P<0.05).2 Neurological function scores of mi R-155 KO group were significantly reduced(P< 0.05). There was no significant difference when added ABL treatment(P > 0.05).3 After TTC staining, knockout(KO) of mi R-155 obviously reduced the cerebral infarct volume induced by MCAO(P< 0.05).4 ABL suppresses mi R-155-mediated inflammatory responses: Western blot analysis showed that the upregulation of TNF-α and IL-1β by MCAO in the ischemic cerebral tissues of mi R-155 KO mice was markedly reduced, and ABL treatment further reduced TNF-α and IL-1β expression in mi R-155 KO mice(P < 0.05). A similar result was obtained by q RT-PCR. We then introduced p Ad-mi R-155 into WT mice through an LV injection to overexpress mi R-155. Western blot analysis showed that mi R-155 overexpression markedly increased the levels of TNF-α and IL-1β of MCAO mice(P < 0.05), and that the upregulation of these two cytokines was slightly reduced by ABL treatment relative to that of mi R-155 overexpression alone. Furthermore, in BV2 microglial cells, overexpression of mi R-155 significantly increased the levels of TNF-α and IL-1β in OGD-treated BV2 cells. Conversely, si RNA-mediated knockdown of mi R-155 was accompanied by a marked reduction in TNF-α and IL-1β compared to the non-silencing control si RNA, and ABL treatment further decreased the expression levels of these two cytokines.Conclusions: mi R-155 expression in ischemic cerebral tissue of MCAO mice and OGD-treated BV2 cells was markedly upregulated. ABL treatment dose-dependently suppressed the expression of mi R-155. ABL suppressed mi R-155-mediated inflammatory responses, reduced TNF-α and IL-1β expression. Part Ⅲ ABL exerts the anti-inflammation action on ischemic cerebraltissues by suppressing mi R-155 expressionObjective: To explore the molecular mechanisms whereby ABL modulates inflammatory response induced by cerebral ischemia.Methods: Male, 8-week-old mi R-155 KO and wild-type(WT) mice were subjected to permanent middle cerebral artery occlusion(p MCAO). p Ad-mi R-155(p Ad as a control) was introduced into WT mice through LV injection to overexpress mi R-155. ABL(20 mg/kg) was injected intraperitoneally 30 minutes prior to MCAO operation. BV2 microglial cells were used in vitro study. Adenovirus infection experiments were performed in BV2 cells to overexpress mi R-155. si RNA transfection experiments were performed in BV2 cells to knock down mi R-155. BV2 cells were treated with or without ABL treatment(100 μmol/L), and then exposed to OGD for 4 hours. Mice were anesthetized and killed at 24 h after stroke. Western blotting and q RT-PCR were used to examine the expression of TLR4, My D88 and SOCS1 in schemic cerebral tissue and BV2 cells. We also examined the levels of p-Akt, p-ERK, p-JNK and p-NF-κB.Results:1 ABL prohibits mi R-155-mediated inflammatory responses by regulating TLR4/My D88 and SOCS1 expression. Western blot analysis showed that marked expression of TLR4 was observed in ischemic cerebral tissue of WT mice at 24 hours after MCAO, and this expression was obviously reduced in mi R-155 KO mice(P<0.05). TLR4 expression was not further decreased in mi R-155 KO mice regardless of ABL treatment. The expression levels of My D88 and SOCS1 were upregulated by ABL in WT mice after MCAO and were further increased in ischemic cerebral tissue of mi R-155 KO mice regardless of ABL treatment. The effect of mi R-155 deletion on TLR4, My D88 and SOCS1 expression was further verified by q RT-PCR of the m RNA expression of these genes. TLR4 expression was significantly increased in ischemic cerebral tissue of mi R-155-overexpressing mice compared to p Ad-infected mice(P < 0.05), and ABL treatment reduced TLR4 expression regardless of whether mi R-155 was overexpressed. Accordingly, mi R-155 overexpression reduced, while ABL treatment increased the expression of My D88 and SOCS1(P < 0.05). In keeping with the in vivo data, mi R-155 overexpression and knockdown in OGD-treated BV2 cells promoted and inhibited, respectively, TLR4 expression, but inhibited and promoted My D88 and SOCS1 expression.2 ABL does not act directly on proinflammatory signaling. Western blotting showed an obvious increase in the levels of p-Akt, p-ERK, p-JNK, and p-NF-κB in ischemic cerebral tissue of mi R-155-overexpressing mice after MCAO compared to p Ad-infected mice(P < 0.05). Conversely, mi R-155 knockout abrogated the activation effect of cerebral ischemia on these signaling molecules. In BV2 microglial cells, ABL treatment did not obviously affect the phosphorylation of Akt, ERK, JNK and p38, as well as NF-κB acetylation induced by OGD.Conclusions: mi R-155 mediates inflammatory responses induced by cerebral ischemia through regulating TLR4/My D88 and SOCS1 expression. ABL exerts its inhibitory action on the activation of proinflammatory signaling cascades by suppressing mi R-155 expression, but it does not act directly on proinflammatory signaling molecules. |
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