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[1]何文全,蘇進益,陸紅日,等.獨活寄生顆粒治療大鼠肩周炎的效果和作用機制研究[J].中醫(yī)正骨,2022,34(06):1-8.
　HE Wenquan,SU Jinyi,LU Hongri,et al.Efficacy and mechanism of Duhuo Jisheng(獨活寄生)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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獨活寄生顆粒治療大鼠肩周炎的效果和作用機制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第34卷
期數(shù):
2022年06期
頁碼:
1-8
欄目:
臨床研究
出版日期:
2022-06-20

文章信息/Info

Title:
Efficacy and mechanism of Duhuo Jisheng(獨活寄生)granules against scapulohumeral periarthritis in rats:an experimental study
作者:
何文全; 蘇進益; 陸紅日; 張洪彬; 賴偉偉
(臺州市中醫(yī)院,浙江 臺州 318001)
Author(s):
HE Wenquan; SU Jinyi; LU Hongri; ZHANG Hongbin; LAI Weiwei
Taizhou Traditional Chinese Medicine Hospital,Taizhou 318001,Zhejiang,China
關(guān)鍵詞:
關(guān)節(jié)周圍炎;  獨活寄生顆粒;  信號傳導(dǎo);  類Toll受體;  髓樣分化因子88;  大鼠;  動物實驗
Keywords:
periarthritis;  Duhuo Jisheng granules;  signal transduction;  toll-like receptors;  myeloid differentiation factor 88;  rats;  animal experimentation
摘要:
目的:探討?yīng)毣罴纳w粒治療大鼠肩周炎的效果和作用機制。方法:從50只大鼠中隨機選取10只作為正常對照組,其余建立肩周炎模型。將建模成功的大鼠隨機分為肩周炎模型組、獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組。獨活寄生顆粒干預(yù)組每天按照3 g·kg-1獨活寄生顆粒進行灌胃,獨活寄生顆粒聯(lián)合脂多糖干預(yù)組每天按照3 g·kg-1獨活寄生顆粒、3 mg·kg-1脂多糖進行灌胃,均以生理鹽水制成5 mL溶液; 正常對照組和肩周炎模型組以5 mL生理鹽水灌胃; 每天灌胃1次,持續(xù)5周。干預(yù)結(jié)束后第1天,進行曠場實驗,并記錄大鼠中央停留時間及活動總路程。曠場實驗結(jié)束后大鼠禁食禁水12 h,處死,切取肩關(guān)節(jié)組織樣本,采用酶聯(lián)免疫吸附分析法檢測肩關(guān)節(jié)組織中腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、白細胞介素(interleukin,IL)-1β、前列腺素E2(prostaglandin E2,PGE2)及5-羥色胺(5-hydroxytryptamine,5-HT)含量,采用免疫印跡法檢測肩關(guān)節(jié)組織中Toll樣受體(Toll-like receptor,TLR)4、TLR2、髓樣分化因子88(myeloid differentiation factor 88,MyD88)蛋白表達量。結(jié)果:①模型建立及分組結(jié)果。成功建立肩周炎模型大鼠32只,肩周炎模型組10只、獨活寄生顆粒干預(yù)組11只、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組11只。②大鼠運動能力評價結(jié)果。4組大鼠中央停留時間、活動總路程比較,組間差異均有統(tǒng)計學(xué)意義[(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]。肩周炎模型組、獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠的中央停留時間均長于正常對照組(LSD-t=26.670,P=0.000; LSD-t=23.143,P=0.000; LSD-t=24.930,P=0.000),活動總路程均短于正常對照組(LSD-t=9.581,P=0.000; LSD-t=4.113,P=0.001; LSD-t=7.017,P=0.000); 獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠的中央停留時間均短于肩周炎模型組(LSD-t=10.385,P=0.000; LSD-t=4.300,P=0.000)、活動總路程均長于肩周炎模型組(LSD-t=5.878,P=0.000; LSD-t=2.984,P=0.008); 獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠中央停留時間長于獨活寄生顆粒干預(yù)組(LSD-t=6.337,P=0.000),活動總路程短于獨活寄生顆粒干預(yù)組(LSD-t=3.024,P=0.000)。③病理學(xué)觀察結(jié)果。HE染色結(jié)果顯示,正常對照組大鼠肩關(guān)節(jié)肌細胞、肌纖維排列整齊,肌組織結(jié)構(gòu)完整,未見增生的毛細血管及炎性細胞; 肩周炎模型組大鼠肩關(guān)節(jié)肌細胞、肌纖維排列混亂,肌組織結(jié)構(gòu)不完整,可見大量增生的毛細血管及炎性細胞; 獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)肌細胞、肌纖維排列及肌組織結(jié)構(gòu)較肩周炎模型組有所改善,增生的毛細血管及炎性細胞較肩周炎模型組少,且獨活寄生顆粒干預(yù)組大鼠肩關(guān)節(jié)肌細胞、肌纖維排列及肌組織結(jié)構(gòu)改善程度大于獨活寄生顆粒聯(lián)合脂多糖干預(yù)組,增生的毛細血管及炎性細胞少于獨活寄生顆粒聯(lián)合脂多糖干預(yù)組。④炎癥相關(guān)指標檢測結(jié)果。4組大鼠肩關(guān)節(jié)組織中TNF-α、IL-1β、PGE2含量比較,組間差異均有統(tǒng)計學(xué)意義[(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)]。肩周炎模型組、獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中TNF-α、IL-1β、PGE2含量均高于正常對照組(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); 獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中TNF-α、IL-1β、PGE2含量均低于肩周炎模型組(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); 獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中TNF-α、IL-1β、PGE2含量均高于獨活寄生顆粒干預(yù)組(LSD-t=2.944,P=0.008; LSD-t=2.954,P=0.008; LSD-t=2.666,P=0.015)。⑤疼痛相關(guān)指標檢測結(jié)果。4組大鼠肩關(guān)節(jié)組織中5-HT含量比較,差異有統(tǒng)計學(xué)意義[(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]。肩周炎模型組、獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中5-HT含量均高于正常對照組(LSD-t=6.813,P=0.000; LSD-t=2.802,P=0.011; LSD-t=4.489,P=0.000); 獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中5-HT含量均低于肩周炎模型組(LSD-t=4.892,P=0.000; LSD-t=2.777,P=0.012); 獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中5-HT含量高于獨活寄生顆粒干預(yù)組(LSD-t=2.105,P=0.048)。⑥炎癥信號通路相關(guān)蛋白檢測結(jié)果。4組大鼠肩關(guān)節(jié)組織中TLR4、TLR2、MyD88蛋白表達量比較,組間差異均有統(tǒng)計學(xué)意義(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)。肩周炎模型組、獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中TLR4、TLR2、MyD88蛋白表達量均高于正常對照組(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); 獨活寄生顆粒干預(yù)組、獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中TLR4、TLR2、MyD88蛋白表達量低于肩周炎模型組(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); 獨活寄生顆粒聯(lián)合脂多糖干預(yù)組大鼠肩關(guān)節(jié)組織中TLR4、TLR2、MyD88蛋白表達量均高于獨活寄生顆粒干預(yù)組(LSD-t=12.603,P=0.000; LSD-t=18.770,P=0.000; LSD-t=17.428,P=0.000)。結(jié)論:采用獨活寄生顆粒治療大鼠肩周炎,能夠緩解疼痛、抑制炎癥反應(yīng),其作用機制可能與抑制TLR/MyD88信號通路相關(guān)蛋白的表達有關(guān)。
Abstract:
Objective:To explore the efficacy of Duhuo Jisheng(獨活寄生,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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通訊作者:賴偉偉 E-mail:[email protected]
更新日期/Last Update: 1900-01-01