The Anti-Inflammatory Effect And Underlying Mechanisms Of Ultra-Low-Molecular-Weight Heparin On Neuro Microglial Cells

BackgroundsExperimental studies in recent years have shown that inflammation is closely related to the occurrence and development of a variety of neurodegenerative diseases. Microglia is a kind of innate immune cell in the brain. Once activated, microglia mediates nerve inflammation through releasing cytokines, pro-inflammatory molecules and reactive oxygen radicals. In the process of inflammatory development, activated microglia synthesizes and releases a variety of immune factors and cytotoxic factors, causes synergies in surrounding glial cells and neurons, and then form feedback system between nerve inflammation and glial cell activation. In this way, the toxicity of each other is gradually enlarged; leading to the formation of chronic inflammation, and eventually leading to the neuronal damage and degeneration, even death.Heparin has anticoagulant, anti-inflammatory, anti-allergic effects. Researchs on disease models have found that, heparin can reduce in vivo inflammation in a variety of diseases. Ultra-low-molecular-weight heparin (ULMWH) is a kind of oligosaccharide fragments obtained by degradation of heparin, which has an average molecular weight of2.2kDa. Compared with heparin, ULMWH has two benefits. First is its blood-brain barrier permeability. Second is its lower incidence of bleeding side effects. So it is suggested to be a potential therapeutic for neurodegenerative diseases. The object of this issue is to study the anti-inflammatory effect to of ULMWH on microglial inflammation and its mechanism.MethodsAnti-inflammatory effects:We use lipopolysaccharide-stimulated murine BV2microglial as established inflammation model. MTT assay was performed to evaluate the cytotoxicity of ULMWH on BV2cells. Nitrate reductase detection was used to examine the impact of ULMWH on NO release in supernatant. We use ELISA assay to measure the effect of ULMWH on inflammatory cytokines release in the supernatant, such as IL-1β and TNF-α. Real-time PCR assay was carried out to measure the changes of mRNA expression of IL-1β, TNF-α and ICAM-1caused by ULMWH. Western blotting analysis was employed to examine the expression of iNOS and COX-2in BV2cells. The changes of intracellular ROS levels caused by ULMWH can be detected by flow cytometry and fluorescence microscopy.Anti-inflammatory mechanisms:Cell immunofluorescence was carried out to observe the influence of ULMWH on NFκB p65subunit nuclear translocation situation in BV2microglia. Western blotting assay was employed to examine the effects of ULMWH on the expression of a series of proteins associated with PI3K/Akt/NFKB signaling pathway (p-Akt, Akt, p-NFκB, NFκB) and MAPKs signaling pathways (p-JNK/JNK, p-ERK/ERK, p-p38/p38).ResultsAnti-inflammatory effects:MTT results showed that ULMWH have no cytotoxic effects on BV2cells. The results of nitrate reductase showed that,50μg/mL of ULMWH can significantly reduce the release of NO in the supernatant. ELISA results showed that, both25μg/mL and50μg/mL of ULMWH could significantly reduce the release of TNF-a in the supernatant. Real-time PCR results showed that, both25μg/mL and50μg/mL of ULMWH could significantly reduce the expression levels of IL-1β and ICAM-1mRNA, only50μg/mL of ULMWH can significantly reduce TNF-a mRNA expression in BV2cells. We use fluorescent probes DCFH-DA to detect intracellular reactive oxygen species level. Observing under a fluorescence microscope, we can find that the green fluorescent of model group cells was significantly enhanced compared with control group, reflecting the significant increase of ROS level. Cells pretreated with25μg/mL or50μg/mL of ULMWH showed significantly decreased green fluorescent compared with model group, reflecting that the intracellular ROS levels were significantly decreased. The experiment results detected by flow cytometry are consistent with the above observations. Western blotting results showed that expression levels of iNOS and COX-2were significantly decreased. Compared with the model group, cells treated with μg/mL or50μg/mL ULMWH could cause the decrease of22.22%or24.44%in iNOS expression and the decline of25.50%or38.64%in COX-2expression.Anti-inflammatory mechanisms:In the results of cell immunofluorescence, nuclear p65protein subunit was labeled by red fluorescent and cell nucleus was labeled by blue fluorescent. In cells pretreated by50μg/mL ULMWH, red fluorescence was clearly separated with blue, reflecting that nuclear transfer situation of p65subunit caused by LPS has been decreased in BV2cells. It means that less amount of p65subunit was transferred into the nucleus. Western blotting results showed that, compared with the model group, the expression of p-Akt and NFκB was reduced, whereas the expression of p-NFκB was elevated in cells pretreated with25μg/mL or50μg/mL ULMWH, reflecting that ULMWH can inhibit Akt phosphorylation and the dephosphorylation of p-NFκB. Western blotting results also showed that, compared with the model group, the expression of p-JNK, p-ERK and p-p38in cells pretreated with25μg/mL or50μg/mL ULMWH were all reduced, reflecting that ULMWH might inhibit the phosphorylation of key proteins in MAPKs pathways. ConclusionUltra-low-molecular-weight heparin (ULMWH) has a strong anti-inflammatory activity in BV2microglia in vitro. By inhibiting Akt phosphorylation, p-NFκB dephosphorylation, nuclear transfer and inhibition of phosphorylation of MAPKs pathway key proteins, it can reduce the expression of COX-2, iNOS, TNF-α, IL-1β and ICAM-1mRNA, and ultimately reduce the level of inflammatory cytokines (TNF-a) and inflammatory mediators (NO and ROS). In a word, ULMWH can inhibit LPS-induced BV2cell inflammation and oxidative stress processes, and then shows protective effect to neuro microglial cells. Therefore, ULMWH has the antagonistic effect to inflammation closely related to the central nervous system neurodegenerative diseases and is a class of potential compound for research.