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摘要
本研究的主要目的是探讨适宜浓度短链脂肪酸（short chain fatty acids，SCFAs）混合物对炎症环境下小胶质细胞的抑炎作用及其机制. 采用脂多糖（LPS）刺激小鼠小胶质细胞系BV-2细胞建立神经炎症模型，并利用CCK8试剂盒检测不同浓度单一的乙酸钠、丙酸钠、丁酸钠处理后的细胞活力. 设计选取这三种SCFAs对细胞活力无影响、且有抑炎效果的特定浓度进行组合（SCFAs mix），进一步检测SCFAs mix对LPS刺激下BV-2细胞炎症反应的影响及机制，包括：a. 用一氧化氮（NO）试剂盒检测NO的释放；b. 用ELISA检测炎症因子TNF-α、IL-6的释放；c. 用qRT-PCR和Western blot检测炎症因子TNF-α、IL-6、炎症小体NLRP3、炎症通路相关蛋白TLR4、NF-κB等的表达变化. 结果表明LPS刺激BV-2细胞4 h后，在体系中添加特定浓度的单一SCFA处理12 h后，不能缓解BV-2细胞的炎症反应，而将上述SCFAs配制成同等终浓度的SCFAs mix处理12 h却能显著降低细胞培养上清液中NO、TNF-α和IL-6（均P<0.001）的量，还能抑制BV-2细胞内iNOS、TNF-α、IL-6和NLRP3 mRNA的升高（均P<0.001）；通过对炎症信号通路关键分子的检测发现，SCFAs mix可以抑制LPS诱导的BV-2细胞内TLR4、MyD88、TRAF6和NF-κB蛋白的表达升高. 综上可见：适宜浓度的混合SCFAs可通过调控TLR4/MyD88/TRAF6/NF-κB炎症通路抑制LPS诱导的小胶质细胞的炎症反应，而起到抗炎的保护作用.
Abstract
To investigate the anti-inflammatory effect and mechanism of proper concentration of short-chain fatty acids (SCFAs) mixture (SCFAs mix) on microglia under inflammatory environment. In this study, the murine microglial cell line BV-2 treated with lipopolysaccharide (LPS) was used as an inflammatory cell model. BV-2 cells were treated with different concentrations of sodium acetate, sodium propionate and sodium butyrate, and SCFAs mix, the cell viability were detected by CCK8 kit. The concentrations of each SCFA and SCFAs mixture (SCFAs mix) will be selected by double standard: having no effect on cell viability and having the anti-inflammation effect. The examination of anti-inflammation effect and its mechanism of SCFAs mix in proper concentration on LPS-stimulated BV-2 cells include: (a) the production of NO of BV-2 cells was detected by NO kit, (b) the inflammatory factors such as TNF-α and IL-6 released into the culture supernatant by BV-2 cells were detected by ELISA, (c) the expression of inflammatory factors such as TNF-α and IL-6, inflammasome NLRP3 and some key factors within inflammatory pathways such as TLR4 and NF-κB, were detected by qRT-PCR and Western-blot. We found that after 4 hours of LPS stimulation on BV-2 cells, the addition of a certain concentration of single SCFA to the system for 12 h could not alleviate the inflammatory response of BV-2 cells, but SCFAs mix with the same terminal concentration of each SCFAs could significantly reduce the levels of NO (P<0.001), TNF-α (P<0.001) and IL-6 (P<0.001) in cell culture supernatants. Simultaneously, SCFAs mix inhibited the increase of iNOS, TNF-α, IL-6 (P<0.001) and inflammasome NLRP3 (P<0.001) mRNA in BV-2 cells induced by LPS. Further study showed that SCFAs mix could inhibit LPS-induced high expression of TLR4, MyD88, TRAF6 and NF-κB proteins, which were the key molecules of the inflammatory signaling pathway in BV-2 cells. In conclusion, proper concentration of SCFAs mix could function as a protective role to inhibit LPS-induced microglial inflammatory responses by regulation of the TLR4/MyD88/TRAF6/NF-κB signaling pathway.
适宜浓度短链脂肪酸混合物对小胶质细胞炎症抑制及机制研
贾雪
近年来，越来越多的研究表明，肠道菌群对宿主的健康有重大影响，它们不但参与调节肠道的稳态，也影响着宿主的脑功
SCFAs主要包括乙酸、丙酸和丁酸. 当中枢神经系统出现障碍时，血脑屏障的通透性增加，SCFAs可以通过血液循环到达脑内，进而影响中枢神经系
小胶质细胞是中枢神经系统的常驻免疫细
目前的研究多基于单一SCFA对小胶质细胞的影响，而在体内是多种SCFAs的协同作用，关于不同浓度SCFAs混合物对小胶质细胞和神经炎症的影响及其作用机制尚不明确. 本研究从特定浓度SCFAs混合物对炎症环境下小胶质细胞的影响着手，研究SCFAs对小胶质细胞炎症反应的作用及相关机制.
1 材料与方法
1.1 主要试剂和仪器
乙酸盐、丙酸盐、丁酸盐和LPS购于Sigma公司；DMEM高糖培养基、双抗、PBS（pH 7.4）和0.05%胰蛋白酶购于Gibco公司；胎牛血清购于Lonsera公司；ECL化学发光试剂和PVDF膜购自Millipore公司；RIPA裂解液和NO检测试剂盒购自碧云天公司；CCK8检测试剂盒购于MedchemExpress公司；TNF-α ELISA试剂盒和 IL-6 ELISA试剂盒购于博士德生物公司；RNA提取试剂盒购于Biomiga公司；qRT-PCR相关试剂购自TAKARA公司；电泳仪购自Bio-Rad公司； NF-κB、TRAF6和MyD88抗体购于CST公司； β-Tubulin、GAPDH、TLR4抗体购于Proteintec公司；低温高速离心机、二氧化碳培养箱、6孔板、12孔板、96孔板和100 mm培养皿购于Thermo公司.
1.2 细胞培养
小鼠BV-2小胶质细胞系购于国家细胞资源共享平台. 细胞培养于含10%胎牛血清和1%双抗的DMEM高糖培养基中，置于37℃，5% C
将BV-2细胞以2×1
细胞分为4组. a. 空白对照组：PBS加入培养体系4 h后，再加入PBS继续处理12 h；b. LPS组：LPS处理BV-2细胞4 h后，向培养体系中加入PBS继续处理12 h；c. LPS+SCFAs mix组：LPS处理BV-2细胞4 h后，向培养体系中加入SCFAs mix继续培养12 h；d. SCFAs mix组：PBS加入培养体系4 h后，再加入SCFAs mix继续处理12 h.
1.3 CCK8法检测细胞活力
向96孔板中每孔加入10 μl的CCK8试剂，置于37℃、5% C
1.4 一氧化氮试剂盒检测细胞上清液NO的含量
收集步骤1.2中4组细胞的培养基上清液，4℃、1 000 r/min、离心5 min收集上清液保存于 －80℃冰箱. 根据试剂盒说明书检测各组细胞上清液NO的含量.
1.5 ELISA法检测BV-2细胞培养上清液中的TNF-α、IL-6的含量
收集上述4组细胞上清，方法同1.4. 参照 TNF-α和IL-6 ELISA试剂盒说明书，稀释标准品，依次为15.6、31.2、62.5、125、250、500、 1000 ng/L，设空白孔、显色液孔和待测样品孔，按一定比例稀释样品后根据说明书进行操作，最后在450 nm处测定各孔吸光度值.
1.6 qRT-PCR检测BV-2细胞内炎症信号的变化
收集上述4组细胞，按照RNA提取试剂盒说明书提取细胞总RNA，然后用Nano-Drop测定RNA的浓度；使用随机引物和PrimeScrip
Table 1 The primer sequences of iNOS,TNF-α,IL-6,NLRP3 and GAPDH
| Gene | Sequences(5'-3') | Length / bp |
|---|---|---|
| iNOS | (F) CCTCCTCCACCCTACCAAGT | 160 |
| (R)CACCCAAAGTGCTTCAGTCA | ||
| TNF-α | (F) CGTCAGCCGATTTGCTATCT | 167 |
| (R)CGGACTCCGCAAAGTCTAAG | ||
| IL-6 | (F) AACGATGATGCACTTGCAGA | 244 |
| (R)GAGCATTGGAAATTGGGGTA | ||
| NLRP3 | (F) ATGCTGCTTCGACATCTCCT | 157 |
| (R)AACCAATGCGAGATCCTGAC | ||
| GAPDH | (F) AGGTCGGTGTGAACGGATTTG | 120 |
| (R) TGTAGACCATGTAGTTGAGGTCA |
1.7 Western blot检测蛋白质表达
收集上述各组细胞，用RIPA裂解液裂解细胞获取蛋白质，通过BCA法检测蛋白质浓度后，进行Western blot检测. 一抗NF-κB（1∶1 000）、TLR4（1∶1 000）、MyD88（1∶1 000）、TRAF6（1∶2 000）、β-Tubulin（1∶1 000）、GAPDH（1∶1 000），二抗IgG羊抗兔、羊抗鼠（1∶4 000）.
1.8 统计学分析
使用SPSS 22.0软件进行统计学分析，多组之间的分析使用单因素方差分析，数据以mean±SEM表示，将P<0.05设定为显著性阈值（*P<0.05，**P<0.01，***P<0.001）.
2 结果
2.1 SCFAs mix浓度设定
我们采用CCK8细胞活力实验检测不同浓度SCFAs对BV-2细胞的毒性，以筛选适宜浓度的SCFAs组合. 结果显示：乙酸钠的浓度梯度设置为5、10、15、20、25 mmol/L，5~20 mmol/L均为不影响细胞活力、无细胞毒性的“安全浓度”， 20 mmol/L乙酸钠为处理12 h和24 h均不影响细胞活力的最高安全浓度（图1a，d）；丙酸钠的浓度梯度设置为1、4、6、8、10 mmol/L，1~8 mmol/L丙酸钠均为安全浓度（图1b）；丁酸钠的浓度梯度设置为0.2、0.6、1.0、1.4、2.0 mmol/L，0.2~ 1 mmol/L均为安全浓度（图1c）. 由于在机体内，丙酸盐和丁酸盐的浓度比例通常是1∶
Fig. 1 Effects of single SCFA with different concentrations and SCFAs mix on the BV-2 cells viability
NOTE: (a, b, c)CCK8 Kit was used to test the toxicity of different concentrations of sodium acetate (NaA), sodium propionate (NaP) and sodium butyrate (NaB) on BV-2 cells after 24 h-treatment. (d)CCK8 Kit was used to test the toxicity of different concentrations of NaA, NaP, NaB and SCFAs mix on BV-2 cells after 12 h-treatment. Statistical comparison by one-way ANOVA with post hoc comparisons of LSD, N=5-6, *P<0.05, **P<0.01, ***P<0.001.
2.2 SCFAs mix可以抑制LPS刺激的BV-2细胞NO的产生
NO的产生可以作为评估小胶质细胞激活程度的指标. 利用NO检测试剂盒检测发现，BV-2细胞上清液中NO的生成量在LPS组显著上升；与之相比，LPS+SCFAs mix组则显著下降（图2a，P<0.001），但单一的乙酸钠（20 mmol/L）、丙酸钠（1 mmol/L）、丁酸钠（1 mmol/L）不能降低LPS刺激BV-2细胞产生的NO（图2a）. 一氧化氮合酶（iNOS）是NO合成的限速酶，其表达也可以间接反映小胶质细胞合成NO的水平. LPS组的BV-2细胞高表达iNOS mRNA，而与LPS组相比，LPS+SCFAs mix组的iNOS mRNA显著下降（图2b， P<0.001）. 表明适宜浓度的SCFAs mix可以削弱LPS诱导的小胶质细胞的激活，具有抑制小胶质细胞介导的炎症反应的效应.
Fig. 2 Effects of SCFAs mix on secretion of NO and the expression of iNOS of BV-2 cells under LPS treatment
NOTE: (a)High release of NO from BV-2 cells into the culture supernatant induced by LPS-treatment was inhibited by SCFAs mix, but not single SCFA. The relative mRNA level was normalized to GAPDH mRNA. (b)High expression of iNOS mRNA in BV-2 cells induced by LPS-treatment was inhibited by SCFAs mix. Statistical comparison by one-way ANOVA with post hoc comparisons of LSD,N=6-8,*P<0.05,**P<0.01, ***P<0.001.
2.3 SCFAs mix可以降低LPS刺激的BV-2细胞内促炎因子和炎症小体的产生
促炎因子（TNF-α和IL-6）和炎症小体（NLRP3）在炎症的产生中起重要作用，是小胶质细胞参与炎症反应的重要介质. 为了研究SCFAs mix是否可以抑制关键炎症介质的产生，我们检测了上述因子的释放和表达情况. ELISA结果显示，与空白组相比，LPS组细胞上清液中的TNF-α和IL-6显著上升（图3a-b，P<0.001）；qRT-PCR结果显示，LPS组BV-2细胞内TNF-α、IL-6和NLRP3在mRNA水平的表达也明显增高（图3c-e，P<0.001）. 而给予SCFAs mix处理（LPS+SCFAs mix组）可以有效抑制TNF-α和IL-6的释放，并降低细胞内TNF-α、IL-6和NLRP3的水平（图3a-e）.
Fig. 3 Effects of SCFAs mix on the secretion or expression of proinflammatory cytokines (TNF-α,IL-6) and the inflammasome (NLRP3) of BV-2 cells under LPS stimulation
NOTE: (a,b)High release of TNF-α and IL-6 protein into the supernatant of BV-2 cells culture medium induced by LPS-stimulation were inhibited by SCFAs mix. The expression of protein of TNF-α and IL-6 were determined by ELISA.(c-e)High expression of TNF-α,IL-6 and NLRP3 mRNA in BV-2 cells induced by LPS-stimulation were inhibited by SCFAs mix. The relative mRNA level was normalized to GAPDH mRNA. Statistical comparison by one-way ANOVA with post hoc comparisons of LSD,N=6,*P<0.05,**P<0.01,***P<0.001.
2.4 SCFAs mix通过抑制小胶质细胞的TLR4-NF-κB通路发挥抑炎作用
为了进一步探究SCFAs mix抑制LPS诱发的BV-2细胞炎症反应的机制，我们通过Western blot检测了BV-2细胞内TLR4炎症通路的重要分子：TLR4、MyD88、TRAF6和NF-κB的表达（图4）. 结果表明，LPS刺激使BV-2细胞内上述分子的表达均显著上升，而当给予SCFAs mix处理后，BV-2细胞内上述分子的表达均下降（图4）. 这表明SCFAs mix可能是通过TLR4-MyD88-TRAF6-NF-κB通路发挥其抑制炎症的作用.
Fig. 4 SCFAs mix alleviates LPS-induced neuroinflammation by suppressing the TLR4/MyD88 /TRAF6/NF-κB signaling pathway
NOTE: (a)Representative Western blot for TLR4, MyD88, TRAF6 and NF-κB expression of BV-2 cells. (b-e)LPS-induced high expression of TLR4(b), MyD88(c), TRAF6(d) and NF-κB(e) were suppressed by SCFAs mix treatment in BV-2 cells. Statistical comparison by one-way ANOVA with post hoc comparisons of LSD, N=6, *P<0.05, **P<0.01, ***P<0.001.
3 讨论
研究表明，在神经炎症相关的神经系统疾病的病理进展中，小胶质细胞的激活可以释放促炎因子，这些促炎因子会加重神经元的损伤、甚至导致神经元的死
SCFAs受体缺陷的无菌小鼠脑内的小胶质细胞存在缺陷，说明SCFAs对小胶质细胞的成熟起重要作
我们的研究以LPS刺激的小胶质细胞BV-2细胞系为体外神经炎症模型，通过细胞活力实验和抑制炎症因子（NO）释放实验寻找可以抑制小胶质细胞炎症反应的SCFAs mix的安全、有效的最佳浓度组合. 结果表明，与LPS组及单一SCFA处理组相比，通过SCFAs mix的处理，可以有效降低LPS刺激BV-2细胞产生的NO，降低细胞内TNF-α、IL-6和炎症小体NLRP3的水平、并降低促炎因子向细胞外的释放，这表明适宜浓度的SCFAs mix对于LPS诱导的小胶质细胞炎症反应有一定的治疗作用. 而不适宜浓度的SCFAs，如在帕金森病和自闭症患者粪便中检测到的SCFAs水平异常，可能是导致神经炎症及神经元死亡的重要因素. 机体适宜浓度及比例的SCFAs是一种对抗神经炎症的保护机制. SCFAs mix的抑炎作用在其他疾病中也有研究：研究人员对炎症相关的结直肠癌模型小鼠喂食乙酸盐、丙酸盐、丁酸盐混合物后发现，结肠组织SCFAs的受体GPR43表达明显升高，同时，肿瘤细胞的增殖受到抑制，结肠TNF-α、IL-6等炎症因子也都明显降低. 因此，喂食SCFAs的抑癌和抑炎作用可能与体内SCFAs浓度升高、从而激活了结肠GPR43受体相
LPS是细菌内毒素，常被用于诱导小胶质细胞的神经炎症模
综上所述，安全、有效的适宜浓度SCFAs mix通过对TLR4-MyD88-TRAF6-NF-κB通路的调控可以抑制LPS刺激的小胶质细胞产生iNOS、TNF-α和IL-6，并且抑制了炎症小体NLRP3的表达，从而发挥抑制小胶质细胞介导的神经炎症的作用；而对体内SCFAs的精确干预可能会对神经炎症有防治意义.
CUI Chun. E-mail: cuichun@jiangnan.edu.cn
SHEN Yan-Qin. E-mail: shenyanqin@jiangnan.edu.cn
