Effects Of Koumine On M1 Polarization In Microglial Cells And Its Relationship With Expression Of Interferon Regulatory Factor 8

Neuropathic pain is a syndrome of chronic pain with the various pathogeneses. The nerve injury or hyperglycemia followed by inflammation involves in the whole process of neuropathic pain. The nerve inflammation is closely related with the activity of microglia in the spinal dorsal horn. According to the different phenotypic function, the activated microglial cells are divided into the two types of microglia, that is, pro-inflammatory M1 microglia and anti-inflammatory M2 microglia. M1 microglia overexpress various M1 markers such as CD68, CD86, TNF-α, IL- 1β and IL-6. We found the glia cells activation in the spinal dorsal horn during the early pathogenesis and progressive phase of the neuropathic pain in our preliminary researchs on the diabetic rats and animal models of the neuropathic pain. And koumine can notably restrain the neuropathic pain resulting from diabetes and peripheral nerve injury, as well as, the activated glial cells of spinal dorsal horn. But how to suppress the activation is not clear. In view of this, we used the microglial in vitro stimulated by LPS and high glucose, and treated with koumine in the non-toxic concentration range to investigate the role of koumine on microglia M1 polarization and its upstream regulatory molecules interferon regulatory factor 8(IRF8) and nuclear factor-kappa B(NF-κB/p65). The study was aimed to preliminarily analyze the molecular mechanism of koumine for M1 polarization inhibition and provide a theoretical basis for the molecular mechanism of it. Objective: To study the effects of koumine on the changes in protein and m RNA expression of M1 markers CD68, CD86, TNF-α, IL-1β, IL-6, IRF8 etc. in BV-2 cells induced by high glucose and LPS. Define the inhibitory effects of koumine on M1 polarization in BV-2 cells and exlore its relationship with the expression of IRF8.Methods: 1. MTT assay was used to screen the conditions for modeling andadministration. Western blot and QPCR assay were used to research the effects of koumine on the changes in the expression of M1 markers CD68, CD86, TNF-α, IL-1β, IL-6 induced by high glucose in BV-2 cells after being pretreated with the koumine of the four different concentrations. 2. We examined the polarization situation of BV-2 cells stimulated by the “classical activation” pathway of LPS. And then we used Western blot and QPCR assay to evaluate the effects of koumine on the variations in the expression of M1 markers CD68, CD86, TNF-α, IL-1β, IL-6 induced by LPS in BV-2 cells after being pretreated with koumine at four different concentrations. 3. Western blot and QPCR assay were used to research the influences of koumine on the diversification in the expression of IRF8 induced by high glucose and LPS in BV-2 cells. The protein level of NF-κB/p65 in high glucose and LPS treated BV-2 cells was measured by Western blot.Result: 1. The rate of viable cells had no significant changes in cells treated with 50 m M high glucose and 1.0 μg/ml LPS for 24 h, comparing with control group. The IC50 of koumine affecting BV-2 for 24 h and 48 h is 1.203 m M and 0.5968 m M respectively. It had no significant effect on the cell viability that 50 m M high glucose or 1 μg/ml LPS was co-incubated with 200 μM koumine for 24 h. High glucose can induce M1 m RNA levels in dose-dependent and time-dependent manner. 2. The result of Western blot assay showed that the protein expression of M1 markers(CD68, CD86, TNF-α, IL-1β, IL-6) were notably enhanced in the high glucose group in contrast to the control. The increasing expression of CD86, TNF-α, IL-6 was significantly suppressed by koumine in a dose dependent manner. When the concentration of koumine exceeded 100 μM, the IL-1β expression was distinctly decreased while it must exceed to 200 μM for CD68. The result of QPCR assay indicated that the m RNA expression of M1 markers were increased after being induced by the high glucose for 24 h, which was inordinately attenuated by the treatment with koumine. The effect was the most significant for CD86 and IL-6. The inhibition was in a dose dependent manner for CD68, TNF-α, IL-1β and the concentration of the strongest suppression was 200 μM. 3. LPS induced the protein and m RNA overexpression of M1 markers. The increasing expression of IL-1β was significantly suppressed by koumine in a dose dependent manner while it mustexceed to 50 μM for CD68, CD86, TNF-α, IL-6. The m RNA expression of CD68, CD86, IL-1β, IL-6 was inordinately reduced by koumine and the drug dose over 50 μM can restrain the expression of TNF-α. 4. The protein and m RNA expression of IRF8 were evidently increased in the high glucose group. The m RNA expression can be obviously inhibited by koumine in various concentrations while 200μM for the protein expression. For NF-κB/p65, Koumine had a modest effect on the protein expression. 5. LPS stimulated the protein and m RNA overexpression of IRF8 which were evidently inhibited by koumine in various concentrations but not the protein level of NF-κB/p65.Conclusion:1. Koumine can suppress M1 polarization induced by high glucose in BV-2 cells. 2. Koumine can suppress M1 polarization induced by LPS in BV-2 cells. 3. Koumine can inhibite the expression of IRF8, upstream molecule of M1 polarization but not NF-κB/p65.