Effect of Simiao Decoction on Vitamin D System and Neutrophil Extracellular Traps in Rats with Rheumatoid Arthritis

doi:10.12114/j.issn.1007-9572.2023.0328

Abstract

Abstract:

Background

As China enters the period of accelerated development of population aging, the proportion of rheumatoid arthritis cases is increasing, causing a serious burden on society and families. The active ingredients in simiao decoction have improved effect, but there is a lack of relevant studies to analyze the action and mechanism of prescriptions.

Objective

To observe the intervention effect of simiao decoction on rats with rheumatoid arthritis established by collageninduced arthritis and its effect on vitamin D system and neutrophil extracellular traps (NETs) .

Methods

From November 2021 to January 2023, 70 SD female rats were selected. The rats were randomly divided into the blank group (control group), model group (model group), methotrexate group (MTX group), vitamin D group (VD group), Simiao decoction low-dose group (SMT-L group), Simiao decoction medium-dose group (SMT-M group) and Simiao decoction high-dose group (SMT-H group) by random number table method, with 10 rats in each group. The rat model of synovial arthritis was established by collagen induction method and the gavage treatment was started 7 d after modeling. The degree of ankle swelling was assessed, and the arthritis index (AI) score of rats in each group the levels of interleukin 6 (IL-6), 25-hydroxyvitamin D₃ [25 (OH) D₃], NETs in serum of rats and the expression levels of 24-hydroxylase (CYP24A1), 1-α hydroxylase (CYP27B1), MPO, vitamin D receptor (VDR) and elastase (NE) in synovial tissue of joint were measured.

Results

The degree of ankle swelling in the control, MTX, VD, SMT-L, SMT-M and SMT-H groups was lower than that in the model group on day 7, the degree of ankle swelling in the model group was higher than the control group, the SMT-M group was higher than the model group on day 14, the degree of ankle swelling in the model group was higher than the control and MTX groups and lower than the VD, SMT-L, SMT-M and SMT-H groups on day 21, the degree of ankle swelling in the control, MTX, VD, SMT-L, SMT-M and SMT-H groups was higher than the model group, and the difference was statistically significant (P<0.05). The AI score of rats in the SMT-H group was lower than that in the model group on day 14, the AI score in the MTX, VD, SMT-L and SMT-H groups was lower than that in the model group on day 21, the score of rats in the MTX, VD, SMT-L, SMT-M, and SMT-H groups was lower than that in the model group on day 28, the difference was statistically significant (P<0.05). Immunohistochemical results showed significant differences in the expression levels of MPO and TNF-α in synovial tissue among the 7 groups (P<0.05), MPO and TNF-α in the model group were higher than those in the control group, while MPO and TNF-α in the MTX, VD and SMT-H groups were lower than those in the model group, TNF-α in the SMT-M group was lower than that in the model group, and the difference was statistically significant (P<0.05). IL-6 and NETs in the model group were higher than those in the control group, and 25 (OH) D₃ was lower than that in the control group. IL-6 and NETs in the MTX, VD and SMT-H groups were lower than those in the model group, and 25 (OH) D₃ was higher than that in the model group. NETs in the SMT-M group were lower than those in the model group, and the difference was statistically significant (P<0.05). CYP24A1 in the model group was higher than that in the control, MTX, VD, SMT-M and SMT-H groups, while CYP27B1 and VDR in the model group were lower than those in the control, MTX, VD, SMT-M and SMT-H groups, MPO was higher than that of the control, MTX, VD, SMT-L, SMT-M and SMT-H groups, and NE was higher than the control, VD, SMT-M and SMT-H groups, and the difference was statistically significant (P<0.05) .

Conclusion

Simiao decoction may inhibit the formation of NETs by regulating the vitamin D system, thereby treating RA.

Key words: Rheumatoid arthritis, Simiao decoction, Vitamin D, Vitamin D system, Neutrophil extracellular traps

摘要：

背景

随着我国进入人口老龄化加速发展期，类风湿关节炎（RA）患病比例不断增加，给社会和家庭造成沉重负担。四妙汤中药物的有效成分确有改善RA的作用，但分析方剂的作用及机制缺乏相关研究。

目的

观察四妙汤对胶原诱导法建立的类风湿关节炎大鼠的干预效果及其对维生素D系统及中性粒细胞胞外诱捕网（NETs）的影响。

方法

2021年11月—2023年1月选取SD雌性大鼠70只，采用随机数字表法将大鼠分为空白组（Control组）、模型组（Model组）、甲氨蝶呤组（MTX组）、维生素D组（VD组）、四妙汤低剂量组（SMT-L组）、四妙汤中剂量组（SMT-M组）、四妙汤高剂量组（SMT-H组），每组10只。通过胶原诱导法建立滑膜型关节炎大鼠模型，于造模7 d后开始灌胃治疗。观察药物干预前后大鼠踝部肿胀程度，测量关节炎指数（AI）评分。采用免疫组化法检测滑膜组织髓过氧化物酶（MPO）、肿瘤坏死因子α（TNF-α）表达情况，检测大鼠血清中白介素6（IL-6）、25-羟基维生素D₃[25（OH）D₃]、NETs水平，检测关节滑膜组织24-羟基化酶（CYP24A1）、1-α羟化酶（CYP27B1）、MPO、维生素D受体（VDR）、弹性蛋白酶（NE）表达水平。

结果

药物干预第第7天Control组、MTX组、VD组、SMT-L组、SMT-M组、SMT-H组踝部肿胀程度低于Model组，第14天Model组踝部肿胀程度高于Control组，SMT-M组踝部肿胀程度高于Model组，第21天Model组踝部肿胀程度高于Control组、MTX组，低于VD组、SMT-L组、SMT-M组、SMT-H组，第28天Control组、MTX组、VD组、SMT-L组、SMT-M组、SMT-H组踝部肿胀程度低于Model组（P<0.05）。第14天SMT-H组大鼠AI评分低于Model组，第21天MTX组、VD组、SMT-L组、SMT-H组AI评分低于Model组，第28天MTX组、VD组、SMT-L组、SMT-M组、SMT-H组AI评分低于Model组（P<0.05）。7组大鼠滑膜组织MPO、TNF-α表达水平比较，差异有统计学意义（P<0.05），其中Model组MPO、TNF-α高于Control组，MTX组、VD组、SMT-H组MPO、TNF-α低于Model组，SMT-M组TNF-α低于Model组（P<0.05）。Model组IL-6、NETs高于Control组，25（OH）D₃低于Control组；MTX组、VD组、SMT-H组IL-6、NETs低于Model组，25（OH）D₃高于Model组；SMT-M组NETs低于Model组（P<0.05）。Model组CYP24A1高于Control组、MTX组、VD组、SMT-M组、SMT-H组，CYP27B1、VDR低于Control组、MTX组、VD组、SMT-M组、SMT-H组，MPO高于Control组、MTX组、VD组、SMT-L组、SMT-M组、SMT-H组，NE高于Control组、VD组、SMT-M组、SMT-H组（P<0.05）。

结论

四妙汤可能通过调控维生素D系统抑制NETs的形成，进而治疗RA。

关键词: 类风湿关节炎, 四妙汤, 维生素D, 维生素D系统, 中性粒细胞胞外诱捕网

LUO Shuman,ZHU Xing,CHEN Shuai, et al. Effect of Simiao Decoction on Vitamin D System and Neutrophil Extracellular Traps in Rats with Rheumatoid Arthritis[J]. Chinese General Practice, 2024, 27(03): 348-356. DOI: 10.12114/j.issn.1007-9572.2023.0328.
罗书曼,朱星,陈帅等. 四妙汤对类风湿关节炎大鼠维生素D系统及中性粒细胞胞外诱捕网的影响研究[J]. 中国全科医学, 2024, 27(03): 348-356. DOI: 10.12114/j.issn.1007-9572.2023.0328.

Figures/Tables 10

References 29

[1]	PRASAD P，VERMA S，SURBHI，et al. Rheumatoid arthritis：advances in treatment strategies[J]. Mol Cell Biochem，2023，478（1）：69-88. DOI：10.1007/s11010-022-04492-3.
[2]	COSTELLO R E，MARSDEN A，MOVAHEDI M，et al. The effect of glucocorticoid therapy on mortality in patients with rheumatoid arthritis and concomitant type II diabetes：a retrospective cohort study[J]. BMC Rheumatol，2020，4：4. DOI：10.1186/s41927-019-0105-4.
[3]	HARRISON S R，LI D Y，JEFFERY L E，et al. Vitamin D，autoimmune disease and rheumatoid arthritis[J]. Calcif Tissue Int，2020，106（1）：58-75. DOI：10.1007/s00223-019-00577-2.
[4]	MYERS L K，WINSTEAD M，KEE J D，et al. 1，25-dihydroxyvitamin D₃ and 20-hydroxyvitamin D₃ upregulate LAIR-1 and attenuate collagen induced arthritis[J]. Int J Mol Sci，2021，22（24）：13342. DOI：10.3390/ijms222413342.
[5]	VERMA S，CHATURVEDI V，GANGULY N K，et al. Vitamin D deficiency：concern for rheumatoid arthritis and COVID-19?[J]. Mol Cell Biochem，2021，476（12）：4351-4362. DOI：10.1007/s11010-021-04245-8.
[6]	PUNCEVICIENE E，GAIZEVSKA J，SABALIAUSKAITE R，et al. Vitamin D and VDR gene polymorphisms' association with rheumatoid arthritis in Lithuanian population[J]. Medicina，2021，57（4）：346. DOI：10.3390/medicina57040346.
[7]	VAN BRUGGEN S，MARTINOD K. The coming of age of neutrophil extracellular traps in thrombosis：where are we now and where are we headed?[J]. Immunol Rev，2023，314（1）：376-398. DOI：10.1111/imr.13179.
[8]	李晓红. 穿心莲内酯通过调节中性粒细胞活性治疗类风湿关节炎的机制研究[D]. 北京：北京中医药大学，2020.
[9]	AGRAZ-CIBRIAN J M，GIRALDO D M，URCUQUI-INCHIMA S. 1，25-Dihydroxyvitamin D3 induces formation of neutrophil extracellular trap-like structures and modulates the transcription of genes whose products are neutrophil extracellular trap-associated proteins：a pilot study[J]. Steroids，2019，141：14-22. DOI：10.1016/j.steroids.2018.11.001.
[10]	HANDONO K，SIDARTA Y O，PRADANA B A，et al. Vitamin D prevents endothelial damage induced by increased neutrophil extracellular traps formation in patients with systemic lupus erythematosus[J]. Acta Med Indones，2014，46（3）：189-198.
[11]	张洪美. 膝骨关节炎的规范诊治与阶梯治疗[J]. 中国骨伤，2019，32（5）：391-395. DOI：10.3969/j.issn.1003-0034.2019.05.001.
[12]	王妍曦，桑希生. 基于《黄帝内经》对"痹"内涵及痹病分类的认识[J]. 中国中医基础医学杂志，2021，27（6）：916-917，1010. DOI：10.19945/j.cnki.issn.1006-3250.2021.06.011.
[13]	于鹰，李玉清. 张灿玾临证方药运用经验[J]. 山东中医药大学学报，2019，43（2）：111-114. DOI：10.16294/j.cnki.1007-659x.2019.02.001.
[14]	张孟之，管连城，秦忠，等. 当归补血汤对维甲酸致骨质疏松模型大鼠卵巢CYP24A1和ERα表达的影响[J]. 中药新药与临床药理，2020，31（5）：502-507. DOI：10.19378/j.issn.1003-9783.2020.05.002.
[15]	何艳新，田乐，吉聪，等. 当归补血汤含药血清对TNF-α诱导HFLS-RA细胞增殖的影响[J]. 中成药，2022，44（4）：1277-1280. DOI：10.3969/j.issn.1001-1528.2022.04.044.
[16]	VITA A A，ALJOBAILY H，LYONS D O，et al. Berberine delays onset of collagen-induced arthritis through T cell suppression[J]. Int J Mol Sci，2021，22（7）：3522. DOI：10.3390/ijms22073522.
[17]	魏伟，吴希美，李元建. 药理实验方法学[M]. 4版. 北京：人民卫生出版社，2010.
[18]	韩宇飞，高明利，刘东武. 类风湿性关节炎的发病机制研究进展综述[J]. 中国卫生标准管理，2021，12（1）：162-165. DOI：10.3969/j.issn.1674-9316.2021.01.061.
[19]	许文亚，刘秋红，李晓茹. 类风湿关节炎从虚论治[J]. 中外医疗，2016，35（34）：195-198. DOI：10.16662/j.cnki.1674-0742.2016.34.195.
[20]	张林华. 基于数据挖掘的中医治疗风湿病临床用药规律与作用机制研究[D]. 北京：北京中医药大学，2019.
[21]	XU K D，MIAO Y，LI P，et al. Licochalcone A inhibits cell growth through the downregulation of the Hippo pathway via PES1 in cholangiocarcinoma cells[J]. Environ Toxicol，2022，37（3）：564-573. DOI：10.1002/tox.23422.
[22]	果彤. 运脾解毒通络祛湿方治疗类风湿关节炎的网络靶标预测及机制初探[D]. 北京：北京中医药大学，2020.
[23]	TANG M S，ZENG Y，PENG W J，et al. Pharmacological Aspects of Natural Quercetin in Rheumatoid Arthritis[J]. Drug Des Devel Ther，2022，16：2043-2053. DOI：10.2147/DDDT.S364759.
[24]	PAN D M，LI N，LIU Y Y，et al. Kaempferol inhibits the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes by blocking activation of the MAPK pathway[J]. Int Immunopharmacol，2018，55：174-182. DOI：10.1016/j.intimp.2017.12.011.
[25]	林婉娜，苏慧琳，李慧敏，等. 黄芪桂枝五物汤抗类风湿性关节炎的作用机制[J]. 中国实验方剂学杂志，2022，28（9）：9-15. DOI：10.13422/j.cnki.syfjx.20220607.
[26]	李倩，吴雍真，管连城，等. 黄芪含药血清对衰老骨髓间充质干细胞CYP24A1，CYP27B1 mRNA和蛋白的影响[J]. 中国实验方剂学杂志，2020，26（17）：49-55. DOI：10.13422/j.cnki.syfjx.20201738.
[27]	张礼节，党万太. 中性粒细胞胞外诱捕网在自身免疫性疾病中的作用及机制研究进展[J]. 现代免疫学，2022，42（6）：536-540.
[28]	WANG W，PENG W C，NING X W. Increased levels of neutrophil extracellular trap remnants in the serum of patients with rheumatoid arthritis[J]. Int J Rheum Dis，2018，21（2）：415-421. DOI：10.1111/1756-185X.13226.
[29]	周琦，孙慧娟，刘树民. 中性粒细胞胞外诱捕网的形成及其在自身免疫性疾病中的作用[J]. 中国中药杂志，2021，46（21）：5568-5575. DOI：10.19540/j.cnki.cjcmm.20210726.702.

组别	只数	第0天	第7天	第14天	第21天	第28天
Control组	10	237.80±16.75	249.00±17.41	270.40±16.75	272.20±17.01	276.60±20.17
Model组	10	240.70±16.26	237.70±18.34	241.70±19.09^a	242.60±20.73^a	243.30±23.33^a
MTX组	10	240.10±15.92	243.40±13.83	259.10±11.40^b	267.50±11.73^b	273.60±11.63^b
VD组	10	232.30±8.93	256.50±12.51^b	267.50±12.47^b	266.00±11.52^b	271.30±7.68^b
SMT-L组	10	236.10±17.62	253.80±20.86^b	262.30±22.31^b	270.60±20.17^b	268.80±21.06^b
SMT-M组	10	236.40±12.08	251.40±12.97	255.00±11.84	263.00±13.10^b	266.60±14.39^b
SMT-H组	10	239.80±14.82	257.90±16.15^b	265.30±16.73^b	271.90±13.83^b	272.70±15.81^b
F值		0.39	1.99	3.52	4.27	4.27
P值		0.88	0.08	<0.05	<0.05	<0.05

组别	只数	第0天	第7天	第14天	第21天	第28天
Control组	10	237.80±16.75	249.00±17.41	270.40±16.75	272.20±17.01	276.60±20.17
Model组	10	240.70±16.26	237.70±18.34	241.70±19.09^a	242.60±20.73^a	243.30±23.33^a
MTX组	10	240.10±15.92	243.40±13.83	259.10±11.40^b	267.50±11.73^b	273.60±11.63^b
VD组	10	232.30±8.93	256.50±12.51^b	267.50±12.47^b	266.00±11.52^b	271.30±7.68^b
SMT-L组	10	236.10±17.62	253.80±20.86^b	262.30±22.31^b	270.60±20.17^b	268.80±21.06^b
SMT-M组	10	236.40±12.08	251.40±12.97	255.00±11.84	263.00±13.10^b	266.60±14.39^b
SMT-H组	10	239.80±14.82	257.90±16.15^b	265.30±16.73^b	271.90±13.83^b	272.70±15.81^b
F值		0.39	1.99	3.52	4.27	4.27
P值		0.88	0.08	<0.05	<0.05	<0.05

组别	只数	第0天	第7天	第14天	第21天	第28天
Control组	10	0.51±0.07	0.44±0.03	0.52±0.04	0.62±0.05	0.87±0.04
Model组	10	2.19±0.10^a	3.02±0.09^a	3.13±0.32^a	3.45±0.23^a	3.98±0.07^a
MTX组	10	2.22±0.09^a	2.80±0.05^b	3.08±0.05	2.52±0.20^b	1.60±0.18^b
VD组	10	2.41±0.13^a	2.81±0.23^b	3.11±0.09	3.68±0.08^b	2.16±0.17^b
SMT-L组	10	2.31±0.11^a	2.66±0.19^b	3.21±0.07	3.79±0.11^b	2.72±0.12^b
SMT-M组	10	2.24±0.16^a	2.55±0.16^b	3.33±0.05^b	3.75±0.06^b	2.42±0.19^b
SMT-H组	10	2.39±0.10^a	2.63±0.14^b	3.16±0.03	3.62±0.05^b	2.11±0.03^b
F值		35.66	35.02	54.72	75.96	50.69
P值		<0.05	<0.05	<0.05	<0.05	<0.05

组别	只数	第0天	第7天	第14天	第21天	第28天
Control组	10	0.51±0.07	0.44±0.03	0.52±0.04	0.62±0.05	0.87±0.04
Model组	10	2.19±0.10^a	3.02±0.09^a	3.13±0.32^a	3.45±0.23^a	3.98±0.07^a
MTX组	10	2.22±0.09^a	2.80±0.05^b	3.08±0.05	2.52±0.20^b	1.60±0.18^b
VD组	10	2.41±0.13^a	2.81±0.23^b	3.11±0.09	3.68±0.08^b	2.16±0.17^b
SMT-L组	10	2.31±0.11^a	2.66±0.19^b	3.21±0.07	3.79±0.11^b	2.72±0.12^b
SMT-M组	10	2.24±0.16^a	2.55±0.16^b	3.33±0.05^b	3.75±0.06^b	2.42±0.19^b
SMT-H组	10	2.39±0.10^a	2.63±0.14^b	3.16±0.03	3.62±0.05^b	2.11±0.03^b
F值		35.66	35.02	54.72	75.96	50.69
P值		<0.05	<0.05	<0.05	<0.05	<0.05

组别	只数	第0天	第7天	第14天	第21天	第28天
Control组	10	0	0	0	0	0
Model组	10	12.30±1.05	12.10±0.87	11.90±0.87	11.70±1.16	11.40±1.07
MTX组	10	12.60±1.83	11.90±1.85	10.50±2.06^a	7.80±1.87^a	5.70±1.41^a
VD组	10	11.90±1.79	11.60±1.71	10.90±1.91	9.70±2.16^a	8.20±1.75^a
SMT-L组	10	12.40±1.17	12.30±1.16	11.10±0.87	10.20±1.13^a	8.50±1.84^a
SMT-M组	10	12.20±1.54	11.70±1.82	11.30±1.33	10.60±1.26	8.90±1.79^a
SMT-H组	10	11.60±2.27	10.90±1.52	9.70±1.70^a	8.30±1.76^a	6.40±2.54^a
F值		89.39	98.59	86.20	68.71	46.92
P值		<0.05	<0.05	<0.05	<0.05	<0.05