Inhibitory effect of IL-27 on the overactivation of microglia
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摘要:
小胶质细胞介导的神经炎症对阿尔茨海默病（Alzheimer’s disease，AD）的发生发展至关重要。通过分析GEO数据库，发现IL-27在AD患者大脑皮质和海马中表达均下降。本研究建立了Aβ1-42损伤BV-2细胞的AD细胞模型、脂多糖（LPS）损伤BV-2细胞的炎症细胞模型和炎症动物模型，给予IL-27以评估其对调节小胶质细胞表型和神经炎症的作用。在动物模型中，通过免疫组化检测海马中Iba1+小胶质细胞数量，通过qPCR、ELISA和Western blot检测TNF-α、IL-1β和IL-6等促炎因子表达水平；在细胞模型中，通过qPCR检测小胶质细胞M1/M2表型标志物的表达水平。为进一步探究IL-27的作用机制，通过Western blot检测给予IL-27和Aβ1-42后小胶质细胞中NF-κB、p-NF-κB、IκBα和p-IκBα的表达水平。研究结果表明，IL-27缓解了LPS诱导的脑内小胶质细胞异常激活，降低TNF-α、IL-1β和IL-6等促炎因子表达水平；将 LPS或Aβ1-42诱导的小胶质细胞从神经毒性的M1型转化为神经保护的M2型，改善了Aβ1-42诱导的细胞内NF-κB和IκBα的异常磷酸化水平。本研究提示IL-27可调控Aβ1-42或LPS诱导下的小胶质细胞M1/M2极化，进而缓解神经炎症。
Abstract:Neuroinflammation mediated by microglia is essential for the occurrence and development of Alzheimer’s disease (AD). Through the analysis of the GEO database, it was found that IL-27 expression decreased in both the cerebral cortex and hippocampus of AD patients. In this study, the AD cell model of BV-2 cells injured by Aβ1-42, the inflammatory cell model of BV-2 cells damaged by LPS, and the inflammatory animal model were established and the effects of IL-27 after its administration in the above models in regulating microglial phenotype and neuroinflammation were evaluated. In the animal models, the number of Iba1+ microglia in the hippocampus was detected by immunohistochemistry, the expression of pro-inflammatory factors such as TNF-α, IL-1β and IL-6 was detected by qPCR, ELISA and Western blot, and the expression of M1/M2 phenotypic markers in microglia was detected by qPCR. To further explore the action mechanism of IL-27, Western blot was used to detect the expression levels of NF-κB, p-NF-κB, IκBα and p-IκBα in microglia after administration of IL-27 and Aβ1-42. The results showed that IL-27 alleviated the abnormal activation of microglia induced by lipopolysaccharide (LPS), decreased the expression of pro-inflammatory factors such as TNF- α, IL-1β and IL-6, transformed microglia induced by LPS or Aβ1-42 from neurotoxic M1 to neuroprotective M2, and improved the abnormal phosphorylation of NF-κB and IκBα induced by Aβ1-42. The research suggested that IL-27 can regulate the M1/M2 polarization of microglia induced by Aβ1-42 or LPS, and alleviate neuroinflammation.
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Keywords:
- Alzheimer’s disease /
- microglia /
- lipopolysaccharide /
- IL-27 /
- polarization
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Figure 1. Analysis of IL-27 expression in the brains of normal and Alzheimer’s disease（AD） patients. In Hippocampus region, control group n=19, AD group n=19. In the entorhinal cortex region, control group n=19, AD group n=15. In the frontal cortex region, control group n=27, AD group n=21. (ns: no significance vs control group)
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Figure 2. Effect of IL-27 on activation of microglia in lipopolysaccharide(LPS)-induced mice brain
A: Expression of Iba1 in the brain of normal mice, LPS-induced inflammation mice, and IL-27 treated mice after LPS-induced inflammation was detected by immunohistochemistry (Scale bar=100 μm); B: Quantitative analysis of Iba1+ levels in A ($\bar{x} $ ± s, n=3, ###P < 0.001 vs control group; **P < 0.01 vs LPS group)
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Figure 3. Effect of IL-27 on the expression of inflammatory factors in the brain of mice induced by LPS ($\bar{x} $ ± s)
A: mRNA levels of Il1b, Il6 and Tnf were detected by RT-qPCR; B: Levels of IL-1β, IL-6, TNF-α were detected by ELISA; C: Representative images of the levels of IL-1β, TNF-α were detected by Western blot; D-E: Quantitative analysis of protein levels of TNF-α/actin and IL-1β/actin(n=5 in ELISA, the other n=3) ###P < 0.001, ##P < 0.01 vs control group; ***P < 0.001, **P < 0.01 vs LPS group
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Figure 4. Effect of IL-27 on mRNA expression of M1/M2 cell markers in LPS-induced microglia ($\bar{x} $ ± s, n=3)
A: mRNA levels of M1-specific makers Il1b, Tnf, Nos2, Nlrp3, Fcgr2b, Cd86 and Fcgr3 were detected by RT-qPCR; B: mRNA levels of M2-specific makers Il10, Chil3 and Arg1 were detected by RT-qPCR ###P < 0.001, ##P < 0.01, #P < 0.05 vs control group; ***P < 0.001, **P < 0.01, *P < 0.05 vs LPS group
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Figure 5. Effect of IL-27 on mRNA expression of M1/M2 cell markers in Aβ1-42-induced microglia ($\bar{x} $ ± s, n=3)
A: mRNA levels of M1-specific makers Il1b, Tnf, Nlrp3 and Nos2 were detected by RT-qPCR; B: mRNA levels M2-specific makers of Il10, Chil3 and Arg1 were detected by RT-qPCR ###P < 0.001, ##P < 0.01, #P < 0.05 vs control group; ***P < 0.001, **P < 0.01, *P < 0.05 vs Aβ1-42 group
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Figure 6. Effect of IL-27 on activation of NF-κB, p-NF-κB, IκBα, and p-IκBα in microglia induced by Aβ1-42
A: Levels of NF-κB, p-NF-κB, IκBα and p-IκBα were detected by Western blot; B-C: Quantitative analysis of protein levels of p-NF-κB/NF-κB and p-IκBα/ IκBα in A (`x ± s, n=3, ###P < 0.001, ##P < 0.01, #P < 0.05 vs control group; ***P < 0.001, **P < 0.01, *P < 0.05 vs Aβ1-42 group)
Table 1 Primer sequences for RT- PCR
Biological indicator Forward primer (5'→3') Reverse primer (5'→3') Tnf TTGGTGGTTTGTGAGTGTGAG GACGTGGAACTGGCAGAAGAG Il6 TTGGTCCTTAGCCACTCCTT TAGTCCTCCTACCCCAATT Il1b ATCTTTTGGGGTCCGTCAACT GCAACTGTTCCTGAACTCAACT Nos2 GTGGACGGGTCGATGTCAC GTTCTCAGCCCAACAATACAAA Nlrp3 ACAAGCCTTTGCTCCAGACCCTAT TGCTCTTCACTGCTATCAAGCCCT Fcgr2b GGGAACCAATCTCGTAGTGTCTGT CCAGAAAGGCCAGGATCTAGTG Cd86 GAGCGGGATAGTAACGCTGA GGCTCTCACTGCCTTCACTC Fcgr3 GTCCAGTTTCACCACAGCCTTC GCCAATGGCTACTTCCACCAC Chil3 GGGCATACCTTTATCCTGAG CCACTGAAGTCATCCATGTC Il10 GGTTGCCAAGCCTTATCGGA ACCTGCTCCACTGCCTTGCT Arg1 GTGAAGAACCCACGGTCTGT CTGGTTGTCAGGGGAGTGTT Actb AGCCATGTACCTAGCCATCC TTTGATGTCACGCACGATTT 
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