Effect of Pricking-bloodletting Therapy Combined with Zhuang-medicine-thread Moxibustion on TLRs/MyD88 Signal Pathway in a Rat Model of Acute Gouty Arthritis

doi:10.12114/j.issn.1007-9572.2022.0775

Abstract

Abstract:

Background

With the change of people's environment and diet structure, acute gouty arthritis (AGA) has become a common clinical disease, which is prone to recurrence, causing harm to patients' health. Pricking-bloodletting therapy combined with Zhuang-medicine-thread moxibustion (moxibustion with a threat prepared with Zhuang herbal medicine) has proven to have a definite therapeutic effect on AGA clinically, but the mechanism of action is not very clear.

Objective

To assess the effect of pricking-bloodletting therapy combined with Zhuang-medicine-thread moxibustion on toll-like receptors /myeloid differentiation factor 88 (TLRs/MyD88) signal pathway in a rat model of AGA to explore the mechanism of action of this treatment in AGA.

Methods

The experiment lasted from May 2021 to March 2022, sixty SD rats were equally randomized into 6 groups: blank group, model group, pricking-bloodletting group, medicated thread group, colchicine group and pricking-bloodletting with medicated thread group. Except for the blank group, the other groups received sodium urate suspension injected into the right ankle cavity to prepare the AGA model. Twenty-four hours after the modelling, colchicine group received intragastric administration of colchicine suspension, pricking-bloodletting group received bloodletting after pricking the Ashi acupoint with a needle, medicated thread group received Zhuang-medicine-thread moxibustion at the site of lesion, and pricking-bloodletting with medicated thread group first received bloodletting after pricking the Ashi acupoint with a needle, then Zhuang-medicine-thread moxibustion at the site of lesion. The swelling degree of the right ankle joint was observed at 6, 12, 24 h and 72 h after modeling. Hematoxylin-eosin staining was used to observe the pathological changes of the synovium of the right ankle joint. The serum levels of interleukin (IL) -10, IL-8 and cyclooxygenase-2 (COX-2) were determined by ELISA. The expressions of MyD88 and IKK-β in the synovium of the right ankle were detected by western blotting.

Results

The transverse diameter of right lateral malleolus in model group, pricking-bloodletting group, medicated thread group or colchicine group was larger than that in blank group at 6, 12, 24, 48 h and 72 h after modeling (P<0.05). The transverse diameter of right lateral malleolus in pricking-bloodletting with medicated thread group was larger than that in blank group at 6, 12, 24 h and 48 h after modeling (P<0.05). The transverse diameter of the right lateral malleolus of the pricking-bloodletting group, medicated thread group, colchicine group or pricking-bloodletting with medicated thread group was smaller than that of the model group at 48 h and 72 h after modeling (P<0.05). Compared with model group, the inflammatory cell infiltration of right ankle in pricking-bloodletting group, medicated thread group, colchicine group and pricking-bloodletting with medicated thread group was improved. The blank group had lower levels of IL-8 and COX-2 and higher level of IL-10 than each of the other 5 groups (P<0.05). The model group had higher levels of IL-8 and COX-2 and lower level of IL-10 than colchicine group, pricking-bloodletting with medicated thread group, pricking-bloodletting group or medicated thread group (P<0.05). The colchicine group had lower levels of IL-8 and COX-2 and higher level of IL-10 than pricking-bloodletting group or medicated thread group (P<0.05). The pricking-bloodletting with medicated thread group had lower levels of IL-8 and COX-2 and higher level of IL-10 than pricking-bloodletting group or medicated thread group (P<0.05). The blank group had lower level of MyD88 than each of the other 5 groups (P<0.05). The blank group had lower level of IKK-β than model group, medicated thread group, pricking-bloodletting group or pricking-bloodletting with medicated thread group (P<0.05). The model group had higher IKK-β level than medicated thread group or colchicine group (P<0.05). The model group had higher MyD8 level than medicated thread group, colchicine group, pricking-bloodletting group or pricking-bloodletting with medicated thread group (P<0.05) .

Conclusion

Pricking-bloodletting with Zhuang-medicine-thread moxibustion is effective in improving the symptoms of AGA by regulating the TLRs/MyD88 signaling pathway, which may be a potential alternative therapy for AGA.

Key words: Arthritis, gouty, Chuang medicine, Zhuang thread moxibustion therapy, Myeloid differentiation factor 88, TLRs/MyD88 signal pathway, Rats

摘要：

背景

随着环境及人们饮食结构的改变，急性痛风性关节炎（AGA）已成为临床常见疾病，AGA易反复发作对患者健康造成危害，临床工作中刺血疗法结合壮医药线点灸对AGA有确切的疗效，但其作用机制尚未明确。

目的

基于Toll样受体（TLRs）/髓样分化因子88（MyD88）信号通路探讨刺血疗法结合壮医药线点灸治疗AGA的作用机制。

方法

实验时间为2021年5月—2022年3月，将60只SD大鼠随机分为空白组、模型组、刺血组、药线组、秋水仙碱组、刺血结合药线组，每组10只。除空白组外，其余各组均将尿酸钠悬浮液注入大鼠右踝关节腔内制备AGA模型，于造模24 h后秋水仙碱组予秋水仙碱悬浮液灌胃治疗，刺血疗法组予阿是穴点刺放血治疗，药线组予壮医药线围灸病灶处，刺血结合药线组先予阿是穴点刺放血再行围灸法。观察造模后6、12、24、72 h右踝关节肿胀程度；苏木素-伊红染色观察右踝关节滑膜组织病理改变；采用酶联免疫吸附试验检测大鼠血清中白介素（IL）-10、IL-8及环氧化酶2（COX-2）水平；蛋白质印迹法检测右踝关节滑膜组织中MyD88和IKK-β蛋白表达。

结果

模型组、刺血组、药线组、秋水仙碱组造模后6、12、24、48、72 h右踝关节外踝横截面直径均高于空白组，刺血结合药线组造模后6、12、24、48 h右踝关节外踝横截面直径高于空白组，刺血组、药线组、秋水仙碱组、刺血结合药线组造模后48、72 h右踝关节外踝横截面直径低于模型组（P<0.05）。刺血组、药线组、秋水仙碱组及刺血结合药线组右踝关节其炎性细胞的浸润情况较模型组有所改善。模型组、刺血组、药线组、秋水仙碱组以及刺血结合药线组大鼠IL-8、COX-2水平高于空白组，IL-10水平低于空白组，秋水仙碱组、刺血结合药线组、刺血组、药线组IL-8、COX-2水平低于模型组，IL-10水平高于模型组，刺血组、药线组大鼠IL-8、COX-2水平高于秋水仙碱组，IL-10水平低于秋水仙碱组，刺血组、药线组大鼠IL-8、COX-2水平高于刺血结合药线组，IL-10水平低于刺血结合药线组（P<0.05）。模型组、药线组、刺血组、刺血结合药线组MyD88、IKK-β水平高于空白组，秋水仙碱组MyD8水平高于空白组，药线组、秋水仙碱组MyD88、IKK-β水平低于模型组，刺血组、刺血结合药线组MyD88水平低于模型组（P<0.05）。

结论

刺血疗法结合壮医药线点灸能通过调节TLRs/MyD88信号通路来改善AGA关节炎性症状，起到治疗作用，刺血疗法结合壮医药线点灸可能是AGA的潜在替代疗法。

关键词: 关节炎，痛风性, 壮族医药学, 壮医药线点灸疗法, 髓样分化因子88, TLRs/MyD88信号通路, 大鼠

ZHU Danmeng,HUANG Yuying,LUO Tong'an, et al. Effect of Pricking-bloodletting Therapy Combined with Zhuang-medicine-thread Moxibustion on TLRs/MyD88 Signal Pathway in a Rat Model of Acute Gouty Arthritis[J]. Chinese General Practice, 2023, 26(20): 2525-2531. DOI: 10.12114/j.issn.1007-9572.2022.0775.
朱丹萌,黄玉莹,罗统安等. 刺血疗法结合壮医药线点灸对急性痛风性关节炎大鼠TLRs/MyD88信号通路的影响[J]. 中国全科医学, 2023, 26(20): 2525-2531. DOI: 10.12114/j.issn.1007-9572.2022.0775.

Figures/Tables 5

References 26

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组别	只数	6 h	12 h	24 h	48 h	72 h
空白组	10	0.71±0.02	0.71±0.03	0.71±0.04	0.72±0.04	0.71±0.04
模型组	10	0.97±0.03^a	1.06±0.07^a	0.97±0.08^a	0.89±0.06^a	0.85±0.05^a
刺血组	10	0.95±0.05^a	1.05±0.06^a	0.94±0.09^a	0.84±0.04^ab	0.80±0.05^ab
药线组	10	0.97±0.07^a	1.08±0.06^a	0.97±0.07^a	0.84±0.06^ab	0.81±0.06^ab
秋水仙碱组	10	0.95±0.06^a	1.04±0.07^a	0.96±0.11^a	0.80±0.05^ab	0.76±0.04^ab
刺血结合药线组	10	0.95±0.07^a	1.08±0.08^a	0.95±0.08^a	0.78±0.05^ab	0.74±0.03^b
F值	F_交互=15.375，F_组间=40.361，F_时间=235.846
P值	P_交互<0.001，P_组间<0.001，P_时间<0.001

组别	只数	6 h	12 h	24 h	48 h	72 h
空白组	10	0.71±0.02	0.71±0.03	0.71±0.04	0.72±0.04	0.71±0.04
模型组	10	0.97±0.03^a	1.06±0.07^a	0.97±0.08^a	0.89±0.06^a	0.85±0.05^a
刺血组	10	0.95±0.05^a	1.05±0.06^a	0.94±0.09^a	0.84±0.04^ab	0.80±0.05^ab
药线组	10	0.97±0.07^a	1.08±0.06^a	0.97±0.07^a	0.84±0.06^ab	0.81±0.06^ab
秋水仙碱组	10	0.95±0.06^a	1.04±0.07^a	0.96±0.11^a	0.80±0.05^ab	0.76±0.04^ab
刺血结合药线组	10	0.95±0.07^a	1.08±0.08^a	0.95±0.08^a	0.78±0.05^ab	0.74±0.03^b
F值	F_交互=15.375，F_组间=40.361，F_时间=235.846
P值	P_交互<0.001，P_组间<0.001，P_时间<0.001

组别	只数	IL-8	IL-10	COX-2
空白组	10	10.39±4.96	58.59±13.49	6.78±4.83
模型组	10	76.06±11.07^a	4.47±2.33^a	62.58±10.16^a
刺血组	10	67.12±9.87^abcd	15.95±11.02^abcd	54.13±8.95^abcd
药线组	10	65.95±12.15^abcd	14.94±10.01^abcd	53.65±9.39^abcd
秋水仙碱组	10	36.27±7.34^ab	40.44±9.75^ab	25.32±6.95^ab
刺血结合药线组	10	30.48±10.62^ab	45.02±6.82^ab	23.87±10.19^ab
F值		71.593	48.553	65.619
P值		<0.001	<0.001	<0.001

组别	只数	IL-8	IL-10	COX-2
空白组	10	10.39±4.96	58.59±13.49	6.78±4.83
模型组	10	76.06±11.07^a	4.47±2.33^a	62.58±10.16^a
刺血组	10	67.12±9.87^abcd	15.95±11.02^abcd	54.13±8.95^abcd
药线组	10	65.95±12.15^abcd	14.94±10.01^abcd	53.65±9.39^abcd
秋水仙碱组	10	36.27±7.34^ab	40.44±9.75^ab	25.32±6.95^ab
刺血结合药线组	10	30.48±10.62^ab	45.02±6.82^ab	23.87±10.19^ab
F值		71.593	48.553	65.619
P值		<0.001	<0.001	<0.001

组别	只数	MyD88	IKK-β
空白组	7	0.31±0.02	0.42±0.02
模型组	7	1.00±0.03^a	0.77±0.05^a
刺血组	7	0.58±0.04^ab	0.64±0.06^a
药线组	7	0.77±0.09^ab	0.58±0.03^ab
秋水仙碱组	7	0.41±0.01^ab	0.45±0.02^b
刺血结合药线组	7	0.53±0.05^ab	0.64±0.08^a
F值		85.941	21.616
P值		<0.001	<0.001