紫草素联合低强度脉冲式超声波调节ERK-JNK/NF-κB信号通路抑制关节炎的药理活性研究

doi:10.16138/j.1673-6087.2024.06.06

摘要/Abstract

摘要：

目的：探讨紫草素（shikonin）对抑制滑膜细胞增殖及炎症反应的可能机制，并评估其联合低强度脉冲式超声波（low-intensity pulsed ultrasound shockwave， LI-PUS）治疗类风湿性关节炎（rheumatoid arthritis， RA）的可行性。方法：体外实验通过脂多糖（lipopolysaccharide， LPS）诱导小鼠滑膜细胞异常增殖。采用细胞计数试剂（cell counting kit-8, CCK-8）和酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测滑膜细胞的增殖情况和炎症因子以及抗氧化物的表达水平。细胞划痕实验检测滑膜细胞的迁移情况。免疫荧光检测滑膜细胞中核因子κB(nuclear factor kappa B, NF-κB)的表达情况。体内实验通过完全弗氏佐剂诱导小鼠RA模型。40只小鼠平均分为正常组、对照组、紫草素组、紫草素+LI-PUS联合治疗组。记录治疗第12天和24天的小鼠关节炎肿胀指数并检测血清中炎症因子水平。蛋白质印迹法（Western blotting）检测小鼠膝关节滑膜组织中c-Jun氨基端蛋白激酶（c-Jun N-terminal protein kinase， JNK）、胞外信号调节激酶（extracellular signal-regulated kinase，ERK）、NF-κB磷酸化水平。结果：紫草素抑制LPS诱导的滑膜细胞炎症细胞因子和氧化应激。在紫草素不同浓度的干预下，滑膜细胞的迁移均被显著抑制，NF-κB蛋白表达含量有所降低。动物实验结果显示，佐剂诱导的关节炎小鼠关节肿胀评分显著升高，紫草素干预后明显降低。紫草素+LI-PUS组治疗效果良好。紫草素抑制炎症细胞因子表达，白介素（interleukin，IL）-1β显著降低，紫草素+LI-PUS联合治疗抗炎作用极显著。蛋白质印迹法结果显示，对照组ERK、JNK、NF-κB磷酸化程度显著高于正常组，紫草素组、紫草素+LI-PUS联合治疗组均显著抑制，且紫草素+LI-PUS联合治疗组优于紫草素组。结论：紫草素可抑制LPS诱导的滑膜细胞异常增殖，且可通过ERK-JNK/NF-κB信号通路发挥抗关节炎活性，中药联合LI-PUS共同治疗优于单独中药治疗。

关键词: 紫草素, 低强度脉冲超声, 关节炎, ERK-JNK/NF-κB

Abstract:

Objective To investigate the possible mechanism of shikonin in inhibiting synovial cell proliferation and inflammatory response and the effect of combining with low-intensity pulsed ultrasound (LI-PUS) in the treatment of rheumatoid arthritis. Methods Abnormal proliferation of mouse synoviocytes were induced by lipopolysaccharide (LPS) in vitro experiments. CCK-8 and ELISA were performed to detect the proliferation of synoviocytes and the expression levels of inflammatory factors and antioxidants. Cell scratch assay was used to detect the migration of synoviocytes. Immunofluorescence was used to detect the expression of nuclear factor (NF)-κB in synoviocytes. In vivo experiments, a mouse rheumatoid arthritis (RA) model was induced by complete Freund’s adjuvant. Forty mice were equally divided into 4 groups, which were normal group, model control group, shikonin perilla treatment group, and shikonin perilla+LI-PUS combination treatment group. The arthritic swelling indexes of the mice were recorded in day 12 and day 24 of treatment. At the end of the experiment, the serum levels of inflammatory factors were tested, and the expression levels of c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and NF-κB in the synovial tissues of the knee joints in mice were detected by Western blot. Results Shikonin inhibited the expression of inflammatory cytokines in LPS-induced synoviocytes. Shikonin intervention was able to significantly reverse LPS-induced oxidative stress in synoviocytes. The migration of synoviocytes were significantly inhibited under the intervention of different concentrations of shikonin. The level of NF-κB protein expression in synovial cells was reduced after the intervention of shikonin. The results of animal experiments showed that adjuvant-induced arthritis mice had significantly higher joint swelling scores, which could be significantly reduced after the intervention of shikonin. Shikonin+LI-PUS group showed good therapeutic effect on mice. Shikonin inhibited the expression of inflammatory cytokines, in which IL-1β was significantly reduced, and the combination of shikonin+LI-PUS showed highly significant anti-inflammatory effect. Western blot results showed that the phosphorylation content of ERK, JNK and NF-κB in the synovial tissue of mice in the model group was significantly higher than that in the normal group, which was significantly suppressed in the shikonin group and the shikonin+LI-PUS combination treatment group, and the shikonin+LI-PUS combination treatment group was superior to the shikonin group. Conclusions Shikonin can inhibit LPS-induced abnormal proliferation of synoviocytes and exert anti-arthritic activity through the ERK-JNK/NF-κB signaling pathway, and the combination of traditional Chinese medicine and low-intensity pulsed ultrasound is superior to the treatment of traditional Chinese medicine alone.

Key words: Shikonin, Low-intensity pulsed ultrasound, Arthritis, ERK-JNK/NF-κB

中图分类号:

R593.2

刘晓旭, 刘金渝, 周本源, 范凯健. 紫草素联合低强度脉冲式超声波调节ERK-JNK/NF-κB信号通路抑制关节炎的药理活性研究[J]. 内科理论与实践, 2024, 19(06): 385-392.

LIU Xiaoxu, LIU Jinyu, ZHOU Benyuan, FAN Kaijian. Research on pharmacological activity of shikonin combined with low-intensity pulsed ultrasound to modulate ERK-JNK/NF-κB signaling pathway to inhibit arthritis[J]. Journal of Internal Medicine Concepts & Practice, 2024, 19(06): 385-392.

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参考文献 22

[1]	Luo R, Su J, Zhang W, et al. Targeted delivery of NO donor and ROS scavenger for synergistic treatment of rheumatoid arthritis[J]. Biomed Pharmacother, 2024, 174:116540.
[2]	Li J, Tang RS, Shi Z, et al. Nuclear factor-κB in rheumatoid arthritis[J]. Int J Rheum Dis, 2020, 23(12):1627-1635.
[3]	Liu C, He L, Wang J, et al. Anti-angiogenic effect of Shikonin in rheumatoid arthritis by downregulating PI3K/AKT and MAPKs signaling pathways[J]. J Ethnopharmacol, 2020, 260:113039.
[4]	Soeken KL, Miller SA, Ernst E. Herbal medicines for the treatment of rheumatoid arthritis: a systematic review[J]. Rheumatology (Oxford), 2003, 42(5):652-659.
[5]	Singh S, Singh TG, Mahajan K, et al. Medicinal plants used against various inflammatory biomarkers for the management of rheumatoid arthritis[J]. J Pharm Pharmacol, 2020, 72(10):1306-1327. doi: 10.1111/jphp.13326 pmid: 32812250
[6]	Liu FY, Wang MQ, Liu MM, et al. Therapeutic effects of shikonin on adjuvant-induced arthritis in rats and cellular inflammation, migration and invasion of rheumatoid fibroblast-like synoviocytes via blocking the activation of Wnt/β-catenin pathway[J]. Phytomedicine, 2023, 116:154857.
[7]	Guo Y, Zhou M, Mu Z, et al. Recent advances in shikonin for the treatment of immune-related diseases: anti-inflammatory and immunomodulatory mechanisms[J]. Biomed Pharmacother, 2023, 165:115138.
[8]	Dai Q, Fang J, Zhang FS. Dual role of shikonin in early and late stages of collagen type Ⅱ arthritis[J]. Mol Biol Rep, 2009, 36(6):1597-1604.
[9]	He L, Luan H, He J, et al. Shikonin attenuates rheumatoid arthritis by targeting SOCS1/JAK/STAT signaling pathway of fibroblast like synoviocytes[J]. Chin Med, 2021, 16(1):96.
[10]	Yang KY, Chen DL. Shikonin inhibits inflammatory response in rheumatoid arthritis synovial fibroblasts via lncRNA-NR024118[J]. Evid Based Complement Alternat Med, 2015, 2015:631737.
[11]	Sun WX, Liu Y, Zhou W, et al. Shikonin inhibits TNF-α production through suppressing PKC-NF-κB-dependent decrease of IL-10 in rheumatoid arthritis-like cell model[J]. J Nat Med, 2017, 71(2):349-356.
[12]	Fu D, Shang X, Ni Z, et al. Shikonin inhibits inflammation and chondrocyte apoptosis by regulation of the PI3K/Akt signaling pathway in a rat model of osteoarthritis[J]. Exp Ther Med, 2016, 12(4):2735-2740. pmid: 27703516
[13]	Zachs DP, Offutt SJ, Graham RS, et al. Noninvasive ultrasound stimulation of the spleen to treat inflammatory arthritis[J]. Nat Commun, 2019, 10(1):951. doi: 10.1038/s41467-019-08721-0 pmid: 30862842
[14]	Prabhakar A, Banerjee R. Nanobubble liposome complexes for diagnostic imaging and ultrasound-triggered drug delivery in cancers: a theranostic approach[J]. ACS Omega, 2019, 4(13):15567-15580. doi: 10.1021/acsomega.9b01924 pmid: 31572858
[15]	Liao AH, Chung HY, Chen WS, et al. Efficacy of combined ultrasound-and-microbubbles-mediated diclofenac gel delivery to enhance transdermal permeation in adjuvant-induced rheumatoid arthritis in the rat[J]. Ultrasound Med Biol, 2016, 42(8):1976-1985.
[16]	Jie SS, Sun HJ, Liu JX, et al. Simiao Yong’an decoction ameliorates murine collagen-induced arthritis by modulating neutrophil activities: an in vitro and in vivo study[J]. J Ethnopharmacol, 2023, 305:116119.
[17]	Pinto BI, Cruz ND, Lujan OR, et al. In vitro scratch assay to demonstrate effects of arsenic on skin cell migration[EB/J]. J Vis Exp, 2019. https://pmc.ncbi.nlm.nih.gov/articles/PMC7537821/.
[18]	Bai Y, Li Y, Marion T, et al. Resistant starch intake alleviates collagen-induced arthritis in mice by modulating gut microbiota and promoting concomitant propionate production[J]. J Autoimmun, 2021, 116:102564.
[19]	Wang M, Su T, Sun H, et al. Regulating Th17/Treg balance contributes to the therapeutic effect of ziyuglycoside i on collagen-induced arthritis[J]. Int J Mol Sci, 2022, 23(24):16105.
[20]	Wang Y, Yan H, et al. An integrated network pharmacology approach reveals that Darutigenol reduces inflammation and cartilage degradation in a mouse collagen-induced arthritis model by inhibiting the JAK-STAT3 pathway[J]. J Ethnopharmacol, 2023, 314:116574.
[21]	Lin Z, Miao J, Zhang T, et al. JUNB-FBXO21-ERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy[J]. Aging Cell, 2021, 20(2):e13306.
[22]	Sun K, Hou L, Guo Z, et al. JNK-JUN-NCOA4 axis contributes to chondrocyte ferroptosis and aggravates osteoarthritis via ferritinophagy[J]. Free Radic Biol Med, 2023, 200:87-101.

紫草素（μmol/mL）	细胞活力（%）	P
0	100.00
5	97.25±1.58	0.955 5
10	95.25±0.69	0.920 6
15	94.25±1.15	0.315 5
20	93.54±1.06	0.077 1
25	91.24±1.38^1）	0.017 7
30	89.26±1.76^2）	0.000 2
35	81.26±1.26^2）	<0.000 1
40	75.25±1.97^2）	<0.000 1
45	73.69±1.68^2）	<0.000 1
50	66.95±1.82^2）	<0.000 1

LPS（ng/mL）	细胞活力（%）	P
0	100.00
1	102.84±1.24	0.999 8
2	105.29±1.18	0.267 2
4	107.84±2.03^1）	0.004 6
8	112.57±1.35^1）	<0.000 1
10	106.88±1.27	0.071 1
20	104.56±1.18	0.090 8

组别	细胞活力（%）	P
正常组	100.00
对照组	114.26±2.19¹⁾	0.001 1
LPS＋紫草素(40 μmol/mL)组	75.69±2.08²⁾	<0.000 1
LPS＋紫草素(50 μmol/mL)组	62.85±1.96²⁾	<0.000 1

组别	IL-1β	IL-6	TNF-α
正常组	74.26±4.29	94.26±5.86	68.57±4.59
对照组	129.86±5.84^1）	138.57±6.85^1）	128.75±7.26^1）
LPS（8 ng/mL）＋紫草素(40 μmol/mL)组	88.96±4.75^2）	104.25±3.96^2）	94.52±5.29^2）
LPS（8 ng/mL）＋紫草素(50 μmol/mL)组	67.85±3.58^2）	82.24±4.56^2）	72.74±4.26^2）

组别	SOD （U/mL）	MDA （nmol/mL）	GSH （μmol/gprot）
正常组	24.26±3.46	21.59±3.69	71.26±4.15
对照组	12.51±4.59^1）	36.26±4.26^1）	42.12±6.23^1）
LPS（8 ng/mL）＋紫草素(40 μmol/mL)组	18.26±4.16^2）	27.96±4.51^2）	57.12±3.96^2）
LPS（8 ng/mL）＋紫草素(50 μmol/mL)组	21.25±3.56^2）	22.86±5.76^2）	67.12±4.29^2）