[1]何文全,苏进益,陆红日,等.独活寄生颗粒治疗大鼠肩周炎的效果和作用机制研究[J].中医正骨,2022,34(06):1-8.
　HE Wenquan,SU Jinyi,LU Hongri,et al.Efficacy and mechanism of Duhuo Jisheng(独活寄生)granules against scapulohumeral periarthritis in rats:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2022,34(06):1-8.
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独活寄生颗粒治疗大鼠肩周炎的效果和作用机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第34卷
期数:
2022年06期
页码:
1-8
栏目:
临床研究
出版日期:
2022-06-20

文章信息/Info

Title:
Efficacy and mechanism of Duhuo Jisheng(独活寄生)granules against scapulohumeral periarthritis in rats:an experimental study
作者:
何文全; 苏进益; 陆红日; 张洪彬; 赖伟伟
(台州市中医院,浙江 台州 318001)
Author(s):
HE Wenquan; SU Jinyi; LU Hongri; ZHANG Hongbin; LAI Weiwei
Taizhou Traditional Chinese Medicine Hospital,Taizhou 318001,Zhejiang,China
关键词:
关节周围炎;  独活寄生颗粒;  信号传导;  类Toll受体;  髓样分化因子88;  大鼠;  动物实验
Keywords:
periarthritis;  Duhuo Jisheng granules;  signal transduction;  toll-like receptors;  myeloid differentiation factor 88;  rats;  animal experimentation
摘要:
目的:探讨独活寄生颗粒治疗大鼠肩周炎的效果和作用机制。方法:从50只大鼠中随机选取10只作为正常对照组,其余建立肩周炎模型。将建模成功的大鼠随机分为肩周炎模型组、独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组。独活寄生颗粒干预组每天按照3 g·kg-1独活寄生颗粒进行灌胃,独活寄生颗粒联合脂多糖干预组每天按照3 g·kg-1独活寄生颗粒、3 mg·kg-1脂多糖进行灌胃,均以生理盐水制成5 mL溶液; 正常对照组和肩周炎模型组以5 mL生理盐水灌胃; 每天灌胃1次,持续5周。干预结束后第1天,进行旷场实验,并记录大鼠中央停留时间及活动总路程。旷场实验结束后大鼠禁食禁水12 h,处死,切取肩关节组织样本,采用酶联免疫吸附分析法检测肩关节组织中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-1β、前列腺素E2(prostaglandin E2,PGE2)及5-羟色胺(5-hydroxytryptamine,5-HT)含量,采用免疫印迹法检测肩关节组织中Toll样受体(Toll-like receptor,TLR)4、TLR2、髓样分化因子88(myeloid differentiation factor 88,MyD88)蛋白表达量。结果:①模型建立及分组结果。成功建立肩周炎模型大鼠32只,肩周炎模型组10只、独活寄生颗粒干预组11只、独活寄生颗粒联合脂多糖干预组11只。②大鼠运动能力评价结果。4组大鼠中央停留时间、活动总路程比较,组间差异均有统计学意义[(5.18±0.52)s,(40.37±4.14)s,(24.81±2.63)s,(33.19±3.51)s,F=253.430,P=0.000;(1 587.43±160.12)cm,(1 008.65±104.17)cm,(1 321.71±135.87)cm,(1 156.08±120.54)cm,F=35.836,P=0.000]。肩周炎模型组、独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠的中央停留时间均长于正常对照组(LSD-t=26.670,P=0.000; LSD-t=23.143,P=0.000; LSD-t=24.930,P=0.000),活动总路程均短于正常对照组(LSD-t=9.581,P=0.000; LSD-t=4.113,P=0.001; LSD-t=7.017,P=0.000); 独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠的中央停留时间均短于肩周炎模型组(LSD-t=10.385,P=0.000; LSD-t=4.300,P=0.000)、活动总路程均长于肩周炎模型组(LSD-t=5.878,P=0.000; LSD-t=2.984,P=0.008); 独活寄生颗粒联合脂多糖干预组大鼠中央停留时间长于独活寄生颗粒干预组(LSD-t=6.337,P=0.000),活动总路程短于独活寄生颗粒干预组(LSD-t=3.024,P=0.000)。③病理学观察结果。HE染色结果显示,正常对照组大鼠肩关节肌细胞、肌纤维排列整齐,肌组织结构完整,未见增生的毛细血管及炎性细胞; 肩周炎模型组大鼠肩关节肌细胞、肌纤维排列混乱,肌组织结构不完整,可见大量增生的毛细血管及炎性细胞; 独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠肩关节肌细胞、肌纤维排列及肌组织结构较肩周炎模型组有所改善,增生的毛细血管及炎性细胞较肩周炎模型组少,且独活寄生颗粒干预组大鼠肩关节肌细胞、肌纤维排列及肌组织结构改善程度大于独活寄生颗粒联合脂多糖干预组,增生的毛细血管及炎性细胞少于独活寄生颗粒联合脂多糖干预组。④炎症相关指标检测结果。4组大鼠肩关节组织中TNF-α、IL-1β、PGE2含量比较,组间差异均有统计学意义[(5.18±0.62)pg·mL-1,(11.23±1.34)pg·mL-1,(7.81±0.91)pg·mL-1,(9.03±1.03)pg·mL-1,F=63.048,P=0.000;(102.23±11.02)pg·mL-1,(153.14±15.71)pg·mL-1,(119.59±12.02)pg·mL-1,(135.76±13.61)pg·mL-1,F=27.584,P=0.000;(185.43±19.01)pg·mL-1,(379.15±39.26)pg·mL-1,(276.71±28.04)pg·mL-1,(310.43±31.21)pg·mL-1,F=71.207,P=0.000)]。肩周炎模型组、独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中TNF-α、IL-1β、PGE2含量均高于正常对照组(TNF-α:LSD-t=12.958,P=0.000; LSD-t=8.145,P=0.000; LSD-t=10.245,P=0.000; IL-1β:LSD-t=8.389,P=0.000; LSD-t=5.126,P=0.000; LSD-t=9.652,P=0.000; PGE2:LSD-t=14.044,P=0.000; LSD-t=12.365,P=0.000; LSD-t=8.335,P=0.000); 独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中TNF-α、IL-1β、PGE2含量均低于肩周炎模型组(TNF-α:LSD-t=6.901,P=0.000; LSD-t=5.746,P=0.000; IL-1β:LSD-t=5.528,P=0.000; LSD-t=16.352,P=0.000; PGE2:LSD-t=6.932,P=0.000; LSD-t=12.687,P=0.000); 独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中TNF-α、IL-1β、PGE2含量均高于独活寄生颗粒干预组(LSD-t=2.944,P=0.008; LSD-t=2.954,P=0.008; LSD-t=2.666,P=0.015)。⑤疼痛相关指标检测结果。4组大鼠肩关节组织中5-HT含量比较,差异有统计学意义[(152.14±24.73)ng·mL-1,(219.58±29.20)ng·mL-1,(180.33±21.37)ng·mL-1,(201.44±25.49)ng·mL-1,F=13.301,P=0.000]。肩周炎模型组、独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中5-HT含量均高于正常对照组(LSD-t=6.813,P=0.000; LSD-t=2.802,P=0.011; LSD-t=4.489,P=0.000); 独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中5-HT含量均低于肩周炎模型组(LSD-t=4.892,P=0.000; LSD-t=2.777,P=0.012); 独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中5-HT含量高于独活寄生颗粒干预组(LSD-t=2.105,P=0.048)。⑥炎症信号通路相关蛋白检测结果。4组大鼠肩关节组织中TLR4、TLR2、MyD88蛋白表达量比较,组间差异均有统计学意义(0.18±0.02,0.89±0.07,0.28±0.03,0.47±0.04,F=520.473,P=0.000; 0.22±0.02,0.91±0.09,0.30±0.03,0.63±0.05,F=353.545,P=0.000; 0.13±0.01,1.15±0.12,0.37±0.04,0.84±0.08,F=386.907,P=0.000)。肩周炎模型组、独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中TLR4、TLR2、MyD88蛋白表达量均高于正常对照组(TLR4:LSD-t=30.840,P=0.000; LSD-t=23.541,P=0.000; LSD-t=21.147,P=0.000; TLR2:LSD-t=23.667,P=0.000; LSD-t=26.985,P=0.000; LSD-t=29.654,P=0.000; MyD88:LSD-t=26.787,P=0.000; LSD-t=30.142,P=0.000; LSD-t=25.333,P=0.000); 独活寄生颗粒干预组、独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中TLR4、TLR2、MyD88蛋白表达量低于肩周炎模型组(TLR4:LSD-t=26.409,P=0.000; LSD-t=20.145,P=0.000; TLR2:LSD-t=21.264,P=0.000; LSD-t=19.874,P=0.000; MyD88:LSD-t=20.393,P=0.000; LSD-t=22.987,P=0.000); 独活寄生颗粒联合脂多糖干预组大鼠肩关节组织中TLR4、TLR2、MyD88蛋白表达量均高于独活寄生颗粒干预组(LSD-t=12.603,P=0.000; LSD-t=18.770,P=0.000; LSD-t=17.428,P=0.000)。结论:采用独活寄生颗粒治疗大鼠肩周炎,能够缓解疼痛、抑制炎症反应,其作用机制可能与抑制TLR/MyD88信号通路相关蛋白的表达有关。
Abstract:
Objective:To explore the efficacy of Duhuo Jisheng(独活寄生,DHJS)granules against scapulohumeral periarthritis(SPA)in rats and its mechanism of action.Methods:Among the 50 rats,10 rats were randomly selected and served as normal control group,and the remained 40 rats were used for inducing SPA.The successfully modeled rats were then randomly assigned to SPA model group,DHJS granules intervention group and DHJS granules combined with lipopolysaccharide(LPS)intervention group.The rats in DHJS granules intervention group were intragastric administrated with DHJS granules in daily dosage of 3 g/kg(DHJS granules were dissolved into 5 mL normal saline(NS)),the ones in DHJS granules combined with LPS intervention group with DHJS granules and LPS in daily dosages of 3 g/kg and 3 mg/kg respectively(DHJS granules and LPS were dissolved into 5 mL NS),and the ones in normal control group and SPA model group with 5 mL NS.All rats in each group were intragastric administration once a day for consecutive 5 weeks.On day 1 after the end of intervention,the open field test was conducted,and the time spent in the central square and total moving distance of the rats were recorded.After the end of open field test,the rats were deprived of food and water for 12 hours,then they were sacrificed and their shoulder tissues were harvested for detecting the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,prostaglandin E2(PGE2)and 5-hydroxytryptamine(5-HT)by using enzyme linked immunosorbent assay(ELISA)and the protein expression levels of Toll-like receptor(TLR)4,TLR2 and myeloid differentiation factor 88(MyD88)by using Western blotting.Results:①Thirty-two SPA rat models were successfully established,10 ones in SPA model group,11 ones in DHJS granules intervention group and DHJS granules combined with LPS intervention group respectively.②There was statistical difference in the time spent in the central square and total moving distance among the 4 groups(5.18±0.52,40.37±4.14,24.81±2.63,33.19±3.51 seconds,F=253.430,P=0.000; 1 587.43±160.12,1 008.65±104.17,1 321.71±135.87,1 156.08±120.54 cm,F=35.836,P=0.000).The time spent in the central square was longer in SPA model group,DHJS granules intervention group and DHJS granules combined with LPS intervention group in contrast to normal control group(LSD-t=26.670,P=0.000; LSD-t=23.143,P=0.000; LSD-t=24.930,P=0.000),and was longer in SPA model group compared to DHJS granules intervention group and DHJS granules combined with LPS intervention group(LSD-t=10.385,P=0.000; LSD-t=4.300,P=0.000),and was longer in DHJS granules combined with LPS intervention group compared to DHJS granules intervention group(LSD-t=6.337,P=0.000).The total moving distance was shorter in SPA model group,DHJS granules intervention group and DHJS granules combined with LPS intervention group in contrast to normal control group(LSD-t=9.581,P=0.000; LSD-t=4.113,P=0.001; LSD-t=7.017,P=0.000),and was shorter in SPA model group compared to DHJS granules intervention group and DHJS granules combined with LPS intervention group(LSD-t=5.878,P=0.000; LSD-t=2.984,P=0.008),and was shorter in DHJS granules combined with LPS intervention group compared to DHJS granules intervention group(LSD-t=3.024,P=0.000).③The HE staining results showed that(1)the regularly arranged muscle cells and muscle fibers as well as the intact muscle tissue structure were observed,while the proliferative capillaries and inflammatory cells were unobserved in rat shoulder tissues of normal control group;(2)the disordered and irregularly arranged muscle cells and muscle fibers as well as the incomplete muscle tissue structure with numerous proliferative capillaries and inflammatory cells were observed in rat shoulder tissues of SPA model group;(3)the arrangement of muscle cells and muscle fibers as well as the structure of muscle tissues were improved,and the proliferative capillaries and inflammatory cells decreased in DHJS granules intervention group and DHJS granules combined with LPS intervention group compared with that of SPA model group; furthermore,the improvement degree was greater,and the proliferative capillaries and inflammatory cells were less in DHJS granules intervention group compared to DHJS granules combined with LPS intervention group.④There was statistical difference in the levels of TNF-α,IL-1β and PGE2 in rat shoulder tissues among the 4 groups(5.18±0.62,11.23±1.34,7.81±0.91,9.03±1.03 pg/mL,F=63.048,P=0.000; 102.23±11.02,153.14±15.71,119.59±12.02,135.76±13.61 pg/mL,F=27.584,P=0.000; 185.43±19.01,379.15±39.26,276.71±28.04,310.43±31.21 pg/mL,F=71.207,P=0.000).The levels of TNF-α,IL-1β and PGE2 in rat shoulder tissues were higher in SPA model group,DHJS granules intervention group and DHJS granules combined with LPS intervention group in contrast to normal control group(TNF-α:LSD-t=12.958,P=0.000; LSD-t=8.145,P=0.000; LSD-t=10.245,P=0.000; IL-1β:LSD-t=8.389,P=0.000; LSD-t=5.126,P=0.000; LSD-t=9.652,P=0.000; PGE2:LSD-t=14.044,P=0.000; LSD-t=12.365,P=0.000; LSD-t=8.335,P=0.000),and were higher in SPA model group compared to DHJS granules intervention group and DHJS granules combined with LPS intervention group(TNF-α:LSD-t=6.901,P=0.000; LSD-t=5.746,P=0.000; IL-1β:LSD-t=5.528,P=0.000; LSD-t=16.352,P=0.000; PGE2:LSD-t=6.932,P=0.000; LSD-t=12.687,P=0.000),and were higher in DHJS granules combined with LPS intervention group compared to DHJS granules intervention group(LSD-t=2.944,P=0.008; LSD-t=2.954,P=0.008; LSD-t=2.666,P=0.015).⑤There was statistical difference in the level of 5-HT in rat shoulder tissues among the 4 groups(152.14±24.73,219.58±29.20,180.33±21.37,201.44±25.49 ng/mL,F=13.301,P=0.000).The level of 5-HT in rat shoulder tissues was higher in SPA model group,DHJS granules intervention group and DHJS granules combined with LPS intervention group in contrast to normal control group(LSD-t=6.813,P=0.000; LSD-t=2.802,P=0.011; LSD-t=4.489,P=0.000),and was higher in SPA model group compared to DHJS granules intervention group and DHJS granules combined with LPS intervention group(LSD-t=4.892,P=0.000; LSD-t=2.777,P=0.012),and was higher in DHJS granules combined with LPS intervention group compared to DHJS granules intervention group(LSD-t=2.105,P=0.048).⑥There was statistical difference in the protein expression levels of TLR4,TLR2 and MyD88 in rat shoulder tissues among the 4 groups(0.18±0.02,0.89±0.07,0.28±0.03,0.47±0.04,F=520.473,P=0.000; 0.22±0.02,0.91±0.09,0.30±0.03,0.63±0.05,F=353.545,P=0.000; 0.13±0.01,1.15±0.12,0.37±0.04,0.84±0.08,F=386.907,P=0.000).The protein expression levels of TLR4,TLR2 and MyD88 in rat shoulder tissues were higher in SPA model group,DHJS granules intervention group and DHJS granules combined with LPS intervention group in contrast to normal control group(TLR4:LSD-t=30.840,P=0.000; LSD-t=23.541,P=0.000; LSD-t=21.147,P=0.000; TLR2:LSD-t=23.667,P=0.000; LSD-t=26.985,P=0.000; LSD-t=29.654,P=0.000; MyD88:LSD-t=26.787,P=0.000; LSD-t=30.142,P=0.000; LSD-t=25.333,P=0.000),and were higher in SPA model group compared to DHJS granules intervention group and DHJS granules combined with LPS intervention group(TLR4:LSD-t=26.409,P=0.000; LSD-t=20.145,P=0.000; TLR2:LSD-t=21.264,P=0.000; LSD-t=19.874,P=0.000; MyD88:LSD-t=20.393,P=0.000; LSD-t=22.987,P=0.000),and were higher in DHJS granules combined with LPS intervention group compared to DHJS granules intervention group(LSD-t=12.603,P=0.000; LSD-t=18.770,P=0.000; LSD-t=17.428,P=0.000).Conclusion:DHJS granules can relieve pain and inhibit inflammatory reaction in rats with SPA,and its mechanism of action may be that it can inhibit the expression of TLR/MyD88 signaling pathway-related proteins.

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