小檗碱联合姜黄素调控M1巨噬细胞极化对动脉粥样硬化的作用机制

doi:10.3969/j.issn.1006-5725.2024.14.003

摘要/Abstract

摘要：

目的探究小檗碱联合姜黄素通过介导巨噬细胞向M1极化对动脉粥样硬化（AS）的作用及机制。方法常规培养小鼠单核巨噬细胞（RAW264.7），通过脂多糖（LPS，100 ng/mL）和γ干扰素（IFN-γ）（20 ng/mL）诱导巨噬细胞RAW264.7获得M1型巨噬细胞，建立细胞模型。将细胞分为对照组、模型组、小檗碱组、姜黄素组、小檗碱+姜黄素联合治疗组。通过CCK-8方法检测小檗碱和姜黄素的处理浓度。ELISA检测各组巨噬细胞上清液M1型巨噬细胞标记物iNOS、TNF-α、CXCL9和p-STAT6/STAT6的表达水平；RT-PCR方法检测iNOS、TNF-α和CXCL9 mRNA水平；Western blot检测各组iNOS、STAT6和p-STAT6蛋白水平。此外，通过siRNA技术下调STAT6水平后，通过Western blot检测p-STAT6蛋白水平的表达；ELISA检测iNOS、TNF-α、CXCL9和p-STAT6表达水平。结果 LPS和IFN-γ联合诱导M1巨噬细胞极化中，相较于其他药物浓度组比，小檗碱（25 μmol/L）、姜黄素（20 μmol/L）为最佳药物浓度，两者单用和联用均不能显著抑制RAW264.7细胞活力（P < 0.05）。与正常组比较，模型组iNOS、TNF-α和CXCL9 mRNA和蛋白水平均升高，p-STAT6/STAT6水平降低，差异有统计学意义（P < 0.05）；与模型组相比，小檗碱组、姜黄素组、小檗碱+姜黄素联合治疗组中iNOS、TNF-α和CXCL9 mRNA和蛋白水平均降低，p-STAT6/STAT6水平升高，且小檗碱+姜黄素联合治疗组中变化的更加明显，差异有统计学意义（P < 0.05）。在小檗碱+姜黄素联合治疗组的M1型巨噬细胞中转染STAT6 siRNA后可下调p-STAT6水平，上调iNOS、TNF-α和CXCL9表达，差异有统计学意义（P < 0.05）。结论小檗碱和姜黄素对 M1型巨噬细胞的活性均有抑制作用，能够减轻炎症反应。此外，小檗碱与姜黄素联合作用较单药使用更有优势，可能是通过活化STAT6发挥其主要作用机制。

关键词: 动脉粥样硬化, M1型巨噬细胞, 小檗碱, 姜黄素, STAT6

Abstract:

Objective To explore the effect and mechanism of berberine combined with curcumin on atherosclerosis （AS） by mediating M1 macrophages polarization. Methods M1-type macrophages were obtained from mouse mononuclear macrophages （RAW264.7） induced by lipopolysaccharide （LPS， 100 ng/mL） and interferon （IFN）-γ （20 ng/mL）. A cell model was established. The cells were divided into a control group， model group， berberine group， curcumin group and berberine plus curcumin group. Concentrations of berberine and curcumin were detected by CCK-8 assay. The expression levels of M1-type macrophage markers iNOS， TNF-α， CXCL9 and p-STAT6/STAT6 in macrophage supernatant were detected by ELISA. Levels of iNOS， TNF-α and CXCL9 mRNA were detected by RT-PCR. Levels of iNOS， STAT6 and p-STAT6 proteins in each group were detected by Western blot. After down-regulation of STAT6 level by siRNA technology， expression of p-STAT6 protein was detected by Western blot. Expression levels of iNOS， TNF-α， CXCL9 and p-STAT6 were detected by ELISA. Results In the polarization of M1 macrophages induced by LPS and IFN-γ， berberine （25 μmol/L） and curcumin （20 μmol/L） were the best concentrations as compared with other drug concentration groups， and neither alone nor combined use could significantly inhibit the viability of RAW264.7 cells （P < 0.05）. As compared with the normal group， iNOS， TNF-α and CXCL9 mRNA and protein levels were increased in the model group， while P-STAT6/STAT6 levels were decreased， with statistical differences （P < 0.05）. As compared with the model group， iNOS， TNF-α and CXCL9 mRNA and protein levels in the berberine group， curcumin group， and berberine plus curcumin group were decreased， while P-STAT6 /STAT6 levels were increased， and the changes were more obvious in berberine plus curcumin group， with statistical difference （P < 0.05）. After transfection of STAT6 siRNA in M1 macrophages in the berberine plus curcumin group， P-STAT6 levels were down-regulated， while expressions of iNOS， TNF-α and CXCL9 were up-regulated， with statistical differences （P < 0.05）. Conclusions Both berberine and curcumin can inhibit the activity of M1-type macrophages and reduce inflammatory response. The action of berberine combined with curcumin is more advantageous than that of either drug alone， which may be the main mechanism of action through activation of STAT6.

Key words: atherosclerosis, M1 macrophage, berberine, curcumin, STAT6

中图分类号:

R543.5

陈玉善,王婷婷,韩心怡,华成俊,靳博远,尚莎莎,宗永华,梁亚州. 小檗碱联合姜黄素调控M1巨噬细胞极化对动脉粥样硬化的作用机制[J]. 实用医学杂志, 2024, 40(14): 1915-1921.

Yushan CHEN,Tingting WANG,Xinyi HAN,Chengjun HUA,Boyuan JIN,Shasha SHANG,Yonghua ZONG,Yazhou. LIANG. Effect of M1 macrophage polarization regulated by berberine combined with curcumin on atherosclerosis[J]. The Journal of Practical Medicine, 2024, 40(14): 1915-1921.

图/表 6

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参考文献 26

1	BLAGOV A V， MARKIN A M， BOGATYREVA A I， et al. The role of macrophages in the pathogenesis of atherosclerosis［J］. Cells， 2023， 12（4）： 522. doi:10.3390/cells12040522 doi: 10.3390/cells12040522
2	HOU P， FANG J， LIU Z， et al. Macrophage polarization and metabolism in atherosclerosis［J］. Cell Death Dis， 2023， 14（10）： 691. doi:10.1038/s41419-023-06206-z doi: 10.1038/s41419-023-06206-z
3	HE Y， LIU T. Oxidized low-density lipoprotein regulates macrophage polarization in atherosclerosis［J］. Int Immunopharmacol， 2023， 120： 110338. doi:10.1016/j.intimp.2023.110338 doi: 10.1016/j.intimp.2023.110338
4	HE P， WANG H， CHENG S， et al. Geniposide ameliorates atherosclerosis by regulating macrophage polarization via perivascular adipocyte-derived CXCL14［J］. J Ethnopharmacol， 2023， 314： 116532. doi:10.1016/j.jep.2023.116532 doi: 10.1016/j.jep.2023.116532
5	ZHOU L， ZHAO H， ZHAO H， et al. GBP5 exacerbates rosacea‐like skin inflammation by skewing macrophage polarization towards M1 phenotype through the NF‐κB signalling pathway［J］. J Eur Acad Dermatol Venereol， 2023， 37（4）： 796-809. doi:10.1111/jdv.18725 doi: 10.1111/jdv.18725
6	HSU C H， PAN Y J， ZHENG Y T， et al. Ultrasound reduces inflammation by modulating M1/M2 polarization of microglia through STAT1/STAT6/PPARγ signaling pathways［J］. CNS Neurosci Ther， 2023， 29（12）： 4113-4123. doi:10.1111/cns.14333 doi: 10.1111/cns.14333
7	GUO H H， SHEN H R， WANG L L， et al. Berberine is a potential alternative for metformin with good regulatory effect on lipids in treating metabolic diseases［J］. Biomed Pharmacother， 2023， 163： 114754. doi:10.1016/j.biopha.2023.114754 doi: 10.1016/j.biopha.2023.114754
8	LAURINDO L F， DE CARBALHO G M， DE OLIVERIRA Zanuso B， et al. Curcumin-Based Nanomedicines in the Treatment of Inflammatory and Immunomodulated Diseases： An Evidence-Based Comprehensive Review［J］. Pharmaceutics， 2023， 15（1）： 229. doi:10.3390/pharmaceutics15010229 doi: 10.3390/pharmaceutics15010229
9	XIONG K， DENG J， YUE T， et al. Berberine promotes M2 macrophage polarisation through the IL-4-STAT6 signalling pathway in ulcerative colitis treatment［J］. Heliyon， 2023，9（3）：e14176. doi:10.1016/j.heliyon.2023.e14176 doi: 10.1016/j.heliyon.2023.e14176
10	ABDOLLAHI E， JOHNSTON TP， GHANEIFAR Z， et al. Immunomodulatory Therapeutic Effects of Curcumin on M1/M2 Macrophage Polarization in Inflammatory Diseases［J］. Curr Mol Pharmacol， 2023，16（1）：2-14. doi:10.2174/1874467215666220324114624 doi: 10.2174/1874467215666220324114624
11	黄勉，李琳，李芬，等. 小檗碱对LPS诱导神经元损伤的作用机制研究［J］. 现代免疫学， 2023，43（6）：465-472.
12	CAO L， YUAN X， ZHANG F E， et al. Effects of curcumin on proliferation and apoptosis and migration of human pterygium fibroblasts［J］. Int Eye Science， 2023： 731-737.
13	HUANG Z， SHEN S， HAN X， et al. Macrophage DCLK1 promotes atherosclerosis via binding to IKKβ and inducing inflammatory responses［J］. EMBO Mol Med， 2023， 15（5）： e17198. doi:10.15252/emmm.202217198 doi: 10.15252/emmm.202217198
14	HUANG H， SUN Z， XU J， et al. Yang-Xin-Shu-Mai granule alleviates atherosclerosis by regulating macrophage polarization via the TLR9/MyD88/NF-κB signaling pathway［J］. J Ethnopharmacol， 2024， 318： 116868. doi:10.1016/j.jep.2023.116868 doi: 10.1016/j.jep.2023.116868
15	JINNOUCHI H， GUO L， SAKAMOTO A， et al. Diversity of macrophage phenotypes and responses in atherosclerosis ［J］. Cell Mol Life Sci， 2020， 77（10）： 1919-1932. doi:10.1007/s00018-019-03371-3 doi: 10.1007/s00018-019-03371-3
16	BARRETT TJ. Macrophages in Atherosclerosis Regression ［J］. Arterioscler Thromb Vasc Biol， 2020， 40（1）： 20-33. doi:10.1161/atvbaha.119.312802 doi: 10.1161/atvbaha.119.312802
17	WEI J， SHEN S， TIAN Y， et al. Correlation analysis of macrophage distribution and pathological features of carotid atherosclerotic plaque［J］. Ann Vasc Surg， 2024，98：355-364. doi:10.1016/j.avsg.2023.08.030 doi: 10.1016/j.avsg.2023.08.030
18	JIAN X， LIU Y， ZHAO Z， et al. The role of traditional Chinese medicine in the treatment of atherosclerosis through the regulation of macrophage activity［J］. Biomed Pharmacother， 2019， 118： 109375. doi:10.1016/j.biopha.2019.109375 doi: 10.1016/j.biopha.2019.109375
19	ADEL-MEHRABAN M S， AGHABEIGLOOEI Z， ATLASI R， et al. Berberine as a Natural Modifier of Gut Microbiota to Promote Metabolic Status in Animal Studies and Clinical Trials： A Systematic Review［J］. Trad Integrat Med， 2023， 2023：202-216. doi:10.18502/tim.v8i2.13086 doi: 10.18502/tim.v8i2.13086
20	DASH R， YADAV M， BISWAL J， et al. Modeling of chitosan modified PLGA atorvastatin-curcumin conjugate （AT-CU） nanoparticles， overcoming the barriers associated with PLGA： An approach for better management of atherosclerosis［J］. Int J Pharm， 2023， 640： 123009. doi:10.1016/j.ijpharm.2023.123009 doi: 10.1016/j.ijpharm.2023.123009
21	LI Z H， CHEN J F， ZHANG J， et al. Mesenchymal stem cells promote polarization of M2 macrophages in mice with acute-on-chronic liver failure via Mertk/JAK1/STAT6 signaling［J］. Stem Cells， 2023， 41（12）： 1171-1184. doi:10.1093/stmcls/sxad069 doi: 10.1093/stmcls/sxad069
22	GERASIMOVA E V， POPKOVA T V， GERASIMOVA D A， et al. Macrophage dysfunction in autoimmune rheumatic diseases and atherosclerosis［J］. Int J of Mol Sci， 2022， 23（9）： 4513. doi:10.3390/ijms23094513 doi: 10.3390/ijms23094513
23	YANG T， WANG R， LIU H， et al. Berberine regulates macrophage polarization through IL-4-STAT6 signaling pathway in Helicobacter pylori-induced chronic atrophic gastritis ［J］. Life Sci， 2021， 266： 118903. doi:10.1016/j.lfs.2020.118903 doi: 10.1016/j.lfs.2020.118903
24	JIANG M， QI Y， HUANG W， et al. Curcumin reprograms TAMs from a protumor phenotype towards an antitumor phenotype via inhibiting MAO-A/STAT6 pathway［J］. Cells， 2022， 11（21）： 3473. doi:10.3390/cells11213473 doi: 10.3390/cells11213473
25	XIONG K， DENG J， YUE T， et al. Berberine promotes M2 macrophage polarisation through the IL-4-STAT6 signalling pathway in ulcerative colitis treatment［J］. Heliyon， 2023，9（3）：e14176. doi:10.1016/j.heliyon.2023.e14176 doi: 10.1016/j.heliyon.2023.e14176
26	AMIRI Z， JALILI S， TARAHOMI M， et al. Curcumin′s spice-infused therapeutic promise： disease severity alleviation in a mouse model of multiple sclerosis via modulation of immune responses［J］. Mol Biol Rep， 2023， 50（11）： 8843-8853. doi:10.1007/s11033-023-08781-y doi: 10.1007/s11033-023-08781-y

目的基因	上游引物序列（5′?3′）	下游引物序列（5′?3′）	扩增片段（bp）
iNOS	CACCACCCTCCTCGTTC	CAATCCACAACTCGCTCC	132
TNF-α	ACGGCATGGATCTCAAAGAC	AGATAGCAAATCGGCTGACG	138
CXCL9	CCGAGGCACGATCCACTACA	CCGGATCTAGGCAGGTTTGA	122
GAPDH	CCTGGAGAAACCTGCCAAGTA	TCATACCAGGAAATGAGCTTGAC	201

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