薯蓣皂苷对滑膜炎大鼠症状的改善作用和对TLR2-NF-κB信号通路的调节作用及其机制

doi:10.13481/j.1671-587X.20210417

摘要/Abstract

摘要： 目的

探讨薯蓣皂苷对滑膜炎大鼠症状的改善作用及对Toll样受体2（TLR2）-核转录因子κB（NF-κB）信号通路的影响，阐明薯蓣皂苷治疗滑膜炎的可能机制。

方法

通过皮内注射弗氏完全佐剂的方法建立滑膜炎大鼠模型，随机分为模型组10只、阳性药组（吲哚美辛10 mg·kg^－1）11只、低剂量薯蓣皂苷（60 mg·kg^－1）组12只和高剂量薯蓣皂苷（120 mg·kg^－1）组12只，另取10只健康SD大鼠设为对照组。采用屈关节疼痛实验评分法和水容积法评价干预后第3、7、14和21天各组大鼠关节疼痛和肿胀情况，酶联免疫吸附测定法检测大鼠滑膜组织中肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）水平，HE染色观察各组大鼠滑膜组织病理形态表现，Western blotting法检测各组大鼠滑膜组织中TLR2、髓样分化因子88（MyD88）、NF-κB和磷酸化NF-κB（p-NF-κB ）p65蛋白表达水平。

结果

与对照组比较，各时间点模型组、阳性药组、低剂量薯蓣皂苷组和高剂量薯蓣皂苷组大鼠疼痛评分明显升高（P<0.05），足趾肿胀程度明显增加（P<0.05），关节滑膜组织中TNF-α和IL-1β水平明显升高（P<0.05）；与模型组比较，各时间点阳性药组、低剂量薯蓣皂苷组和高剂量薯蓣皂苷组疼痛评分明显降低（P<0.05），足趾肿胀程度明显降低（P<0.05），关节滑膜组织中TNF-α和IL-1β水平明显降低（P<0.05）；与阳性药组比较，各时间点低剂量薯蓣皂苷组和高剂量大鼠薯蓣皂苷组疼痛评分明显升高（P<0.05），足趾肿胀程度明显增加（P<0.05），关节滑膜组织中TNF-α和IL-1β水平明显升高（P<0.05）；与低剂量薯蓣皂苷组比较，各时间点高剂量薯蓣皂苷组大鼠疼痛评分明显降低（P<0.05），足趾肿胀程度明显降低（P<0.05），关节滑膜组织中TNF-α和IL-1β水平明显降低（P<0.05）。HE染色，对照组大鼠滑膜组织形态正常，细胞排列整齐，无水肿、充血和增生，未见炎症细胞浸润；模型组大鼠滑膜组织水肿、充血严重，滑膜细胞排列紊乱并大量增生，大量炎症细胞浸润；低剂量薯蓣皂苷组大鼠滑膜组织充血、水肿减轻，细胞增生略有减少；阳性药组和高剂量薯蓣皂苷组大鼠滑膜组织轻度水肿、充血，细胞排列相对整齐，少量炎症细胞浸润，少见增生。与对照组比较，模型组、阳性药组、低剂量薯蓣皂苷组和高剂量薯蓣皂苷组大鼠滑膜组织中TLR2、MyD88和p-NF-κB p65蛋白表达水平明显升高（P<0.05）；与模型组比较，阳性药组、低剂量薯蓣皂苷组和高剂量薯蓣皂苷组大鼠滑膜组织中TLR2、MyD88和p-NF-κB p65蛋白表达水平明显降低（P<0.05）；与阳性药组比较，低剂量薯蓣皂苷组和高剂量薯蓣皂苷组大鼠滑膜组织中TLR2、MyD88和p-NF-κB p65蛋白表达水平明显升高（P<0.05）；与低剂量薯蓣皂苷组比较，高剂量薯蓣皂苷组大鼠滑膜组织中TLR2、MyD88和p-NF-κB p65蛋白表达水平明显降低（P<0.05）。

结论

薯蓣皂苷可能通过抑制TLR2-NF-κB信号通路改善滑膜炎大鼠症状。

关键词: 薯蓣皂苷, 滑膜炎, Toll样受体2, 核转录因子kappa B, 炎症细胞因子

Abstract: Objective

To investigate the improvement effect of diosgenin on the symptoms of synovitis rats and the influence in Toll-like receptor 2 （TLR2） - nuclear factor kappaB（NF-κB） signaling pathway， and to elucidate the possible mechanism of diosgenin in the treatment of synovitis.

Methods

The synovitis models were established by intradermal injection of Freund’s complete adjuvant，and the model rats were randomly divided into model group（n=10），positive drug group （indomethacin 10 mg·kg^－1）（n=11）， low dose of diosgenin group （60 mg·kg^－1）（n=12）， and high dose of diosgenin group （120 mg·kg^－1）（n=12）； another 10 healthy rats were set as control group. Joint pain scoreing and water volume methods were used to evaluate the changes of joint pain and swelling on the 3rd， 7th， 14th and 21st days after intervention. The levels of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） in synovial tissue of the rats in various groups were detected by enzyme-linked immunosorbent assay method. HE staining was used to observe the pathomorphology of synovium tissue of the rats in various groups. Western bloting method was used to detect the expression levels of TLR2， MyD88， NF-κB and p-NF-κB p65 proteins in synovium tissue of the rats in various groups.

Results

Compared with control group， the pain scores of the rats in model group， positive drug group， low dose of diosgenin group and high dose of diosgenin group at different time points were significantly increased P<0.05）， the degrees of toe swelling were significantly increased （P<0.05）， and the levels of TNF-α and IL-1β in synovial tissue were significantly increased （P<0.05）. Compared with model group， the pain scores of the rats in positive drug group， low dose of diosgenin group and high dose of diosgenin group at different time points were significantly decreased （P<0.05）， the degrees of toe swelling were significantly decreased （P<0.05）， and the levels of TNF-α and IL-1β in synovial tissue were significantly decreased （P<0.05）.Compared with positive drug group， the pain scores of the rats in low dose of diosgenin group and high dose of diosgenin group at different time points were significantly increased （P<0.05）， the degrees of toe swelling were significantly increased （P<0.05），and the levels of TNF-α and IL-1β in synovial tissue were significantly increased （P<0.05）. Compared with low dose of diosgenin group， the pain score of the rats in high dose of diosgenin group was significantly decreased（P<0.05）， the degree of toe swelling was significantly decreased （P<0.05）， and the levels of TNF-α and IL-1β in synovial tissue were significantly decreased （P<0.05）. The results of HE staining showed that the synovial tissue of the rats in control group was normal， the cells arranged orderly， without edema， hyperemia and proliferation， and had no inflammatory cell infiltration； the synovial tissue of the rats in model group had serious edema and hyperemia，the synovial cells were disordered and proliferaed， and there was a large number of inflammatory cell infiltration； the hyperemia and edema of the synovial tissue of the rats in low dose of diosgenin group were alleviated， and the cell proliferation was slightly reduced； in positive drug group and high dose of diosgenin group， the synovial tissue was slightly edema and congestive， the cells were regularly arranged，and there was small amount of inflammatory cell infiltration and rare proliferation. Compared with control group， the expression levels of TLR2， MyD88 and p-NF-κB p65 proteins in synovial tissue of the rats in model group， positive drug group， low dose of diosgenin group and high dose of diosgenin group were significantly increased （P<0.05）；compared with model group， the expression levels of TLR2， MyD88 and p-NF-κB p65 proteins in synovial tissue of the rats in positive drug group， low dose of diosgenin group and high dose of diosgenin group were significantly increased （P<0.05）；compared with positive group， the expression levels of TLR2， MyD88 and p-NF-κB p65 proteins in synovial tissue of the rats in low and high doses of diosgenin groups were significantly increased （P<0.05）； compared with low dose of diosgenin group， the expression levels of TLR2， MyD88 and p-NF-κB p65 proteins in synovial tissue of the rats in high dose of diosgenin group were significantly decreased（P<0.05）.

Conclusion

Diosgenin may improve the symptoms of synovitis rats by inhibiting the TLR2-NF- κB signaling pathway.

Key words: diosgenin, synovitis, Toll-like receptor 2, nuclear factor-κB, inflammatory cytokines

中图分类号:

R285.5

武豪杰,张明辉,洪成智. 薯蓣皂苷对滑膜炎大鼠症状的改善作用和对TLR2-NF-κB信号通路的调节作用及其机制[J]. 吉林大学学报(医学版), 2021, 47(4): 943-950.

Haojie WU,Minghui ZHANG,Chengzhi HONG. Improvement effect of diosgenin on symptoms of synovitis rats and its reglatory effect on TLR2-NF-κB signaling pathway and mechanism[J]. Journal of Jilin University(Medicine Edition), 2021, 47(4): 943-950.

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Group	n	Pain score
Group	n	（t/d） 3	7	14	21
Control	10	0.64±0.11	0.66±0.12	0.61±0.14	0.65±0.13
Model	10	10.62±1.23^*	12.98±1.16^*□	14.78±1.45^*□▲	16.69±1.98^*□▲●
Positive drug	11	7.14±0.75^*△	5.01±1.52^*△□	3.58±0.74^*△□▲	1.78±0.35^{*△□▲●}
Diosgenin Low dose	12	9.42±1.27^*△#	8.39±1.58^*△#□	7.39±0.71^*△#□▲	4.74±0.45^{*△#□▲●}
High dose	12	8.25±1.28^*△#O	6.77±1.54^*△#O□	4.94±0.77^*△#O□▲	2.78±0.29^{*△#O□▲●}
F		140.339	115.942	395.789	542.709
P		<0.01	<0.01	<0.01	<0.01

Group	n	Toe swelling degree
Group	n	（t/d） 3	7	14	21
Control	10	0.03±0.01	0.04±0.01	0.02±0.01	0.03±0.01
Model	10	0.75±0.08^*	0.97±0.09^*□	1.45±0.19^*□▲	1.61±0.09^*□▲●
Positive drug	11	0.41±0.05^*△	0.32±0.04^*△□	0.24±0.03^*△□▲	0.12±0.02^{*△□▲●}
Diosgenin Low dose	12	0.61±0.07^*△#	0.48±0.08^*△#□	0.37±0.04^*△#□▲	0.24±0.05^{*△#□▲●}
High dose	12	0.53±0.08^*△#○	0.37±0.04^*△#○□	0.29±0.05^{*△#○□▲}	0.19±0.03^{*△#○□▲●}
F		182.726	326.404	418.313	1 920.000
P		<0.01	<0.01	<0.01	<0.01

Group	n	TNF-α	IL-1β
Control	10	61.22±7.03	5.26±0.24
Model	10	180.39±10.06^*	10.47±0.95^*
Positive drug	11	139.25±11.02^*△	5.31±0.53^*△
Diosgenin Low dose	12	104.34±8.04^*△#	8.44±0.74^*△#
High dose	12	90.29±9.03^*△#○	6.98±0.85^*△#○
F		259.719	99.563
P		<0.01	<0.01

Group	n	TLR2	MyD88	NF-κB	p-NF-κB p65
Control	10	0.24±0.06	0.17±0.04	0.59±0.12	0.05±0.01
Model	10	0.85±0.09^*	0.69±0.07^*	0.58±0.15	0.45±0.08^*
Positive drug	11	0.33±0.04^*△	0.32±0.08^*△	0.60±0.14	0.11±0.02^*△
Diosgenin Low dose	12	0.49±0.08^*△#	0.48±0.09^*△#	0.61±0.12	0.36±0.05^*△#
High dose	12	0.37±0.05^{*△# ○}	0.39±0.11^*△#○	0.59±0.11	0.19±0.03^*△#○
F		41.877	54.856	0.088	151.553
P		<0.01	<0.01	0.986	<0.01