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[1]何曉娟,林平冬,馬玉環(huán),等.獨活寄生湯含藥血清抑制白細胞介素1β誘導(dǎo)的軟骨細胞炎癥反應(yīng)的作用機制研究[J].中醫(yī)正骨,2017,29(08):1-7.
　HE Xiaojuan,LIN Pingdong,MA Yuhuan,et al.Study on mechanism of action of Duhuo Jisheng Tang(獨活寄生湯)medicated serum in inhibiting inflammatory reaction induced by interleukin-1 beta in chondrocytes[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(08):1-7.
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獨活寄生湯含藥血清抑制白細胞介素1β誘導(dǎo)的軟骨細胞炎癥反應(yīng)的作用機制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期數(shù):
2017年08期
頁碼:
1-7
欄目:
基礎(chǔ)研究
出版日期:
2017-08-20

文章信息/Info

Title:
Study on mechanism of action of Duhuo Jisheng Tang(獨活寄生湯)medicated serum in inhibiting inflammatory reaction induced by interleukin-1 beta in chondrocytes
作者:
何曉娟1; 林平冬1; 馬玉環(huán)1; 翁霞萍1; 鄭文偉1; 李西海1; 林潔2; 付長龍2; 邵翔2; 葉蕻芝2; 劉獻祥1
1.福建中醫(yī)藥大學(xué),福建 福州 350122; 2.福建省中西醫(yī)結(jié)合老年性疾病重點實驗室,福建 福州 350122
Author(s):
HE Xiaojuan1; LIN Pingdong1; MA Yuhuan1; WENG Xiaping1; ZHENG Wenwei1; LI Xihai1; LIN Jie2; FU Changlong2; SHAO Xiang2; YE Hongzhi2; LIU Xianxiang1
1.Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 2.Fujian Key Laboratory of Integrated Medicine on Geriatrics,Fuzhou 350122,Fujian,China
關(guān)鍵詞:
骨關(guān)節(jié)炎;  軟骨細胞;  獨活寄生湯;  軟骨退變;  G蛋白偶聯(lián)信號通路;  白細胞介素1β;  基質(zhì)金屬蛋白酶13;  炎癥反應(yīng);  動物實驗
Keywords:
Key words osteoarthritis;  chondrocytes;  Duhuo Jisheng Tang;  cartilage degeneration;  G protein coupled signaling pathway;  interleukin-1beta;  matrix metalloproteinase 13;  inflammatory reaction;  animal experimentation
摘要:
目的:探討?yīng)毣罴纳鷾幯逡种瓢准毎樗?β(interleukin-1 beta,IL-1β)誘導(dǎo)的軟骨細胞炎癥反應(yīng)的作用機制。方法:將10只2月齡雄性SD大鼠隨機分為正常組和獨活寄生湯組,獨活寄生湯組以9.3 g·kg-1劑量的獨活寄生湯灌胃,正常組給予等量生理鹽水灌胃; 每日灌胃2次,連續(xù)1周; 末次灌胃后,經(jīng)腹主動脈取血,分別制備獨活寄生湯含藥血清及空白血清,低溫保存?zhèn)溆谩＝厝?只4周齡SD大鼠膝關(guān)節(jié)軟骨,采用酶消化法分離并培養(yǎng)軟骨細胞,光學(xué)顯微鏡下觀察軟骨細胞形態(tài),并用Ⅱ型膠原酶免疫組化鑒定。取生長良好的第2代軟骨細胞,采用MTT法檢測含藥血清培養(yǎng)24 h、36 h、48 h、72 h后的軟骨細胞活性,采用酶聯(lián)免疫吸附法測定不同濃度IL-1β干預(yù)后軟骨細胞的基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)-13含量。將培養(yǎng)好的第2代軟骨細胞隨機分為空白血清組、模型組、獨活寄生湯含藥血清組,其中空白血清組以含10%空白血清的培養(yǎng)基(dulbecco modified eagle medium,DMEM)培養(yǎng); 模型組加入濃度為15 ng·mL-1的IL-1β干預(yù)24 h后,采用含10%空白血清的DMEM培養(yǎng); 獨活寄生湯含藥血清組加入濃度為15 ng·mL-1的IL-1β干預(yù)24 h后,采用含10%獨活寄生湯含藥血清的DMEM培養(yǎng); 3組均連續(xù)培養(yǎng)48 h,采用Western blot法檢測軟骨細胞中G蛋白偶聯(lián)信號傳導(dǎo)系統(tǒng)關(guān)鍵調(diào)控因子的蛋白表達情況。結(jié)果:①軟骨細胞形態(tài)及免疫組化鑒定結(jié)果。第2代軟骨細胞胞漿豐富,細胞周圍可見具有折光性的細胞外基質(zhì),細胞長勢呈“鋪路石”狀,胞漿呈棕黃色陽性染色。②獨活寄生湯含藥血清培養(yǎng)不同時間后的軟骨細胞活性。含藥血清培養(yǎng)24 h、36 h、48 h、72 h后的軟骨細胞活性比較,差異有統(tǒng)計學(xué)意義(1.01±0.01,1.03±0.02,1.07±0.01,1.02±0.02,F=8.300,P=0.008)。含藥血清培養(yǎng)24 h時的軟骨細胞活性低于培養(yǎng)48 h時的軟骨細胞活性(t=-4.648,P=0.002); 與培養(yǎng)36 h、72 h時的軟骨細胞活性比較,差異無統(tǒng)計學(xué)意義(t=-1.549,P=0.160; t=-0.775,P=0.461)。③不同濃度IL-1β干預(yù)后的MMP-13含量。在含IL-1β濃度為0 ng·mL-1、5 ng·mL-1、10 ng·mL-1、15 ng·mL-1、20 ng·mL-1、25 ng·mL-1的DMEM中培養(yǎng)24 h后軟骨細胞MMP-13含量比較,差異有統(tǒng)計學(xué)意義[(0.07±0.01)ng·mL-1,(0.08±0.01)ng·mL-1,(0.09±0.01)ng·mL-1,(0.10±0.01)ng·mL-1,(0.08±0.01)ng·mL-1,(0.06±0.01)ng·mL-1,F=6.823,P=0.001]。未經(jīng)IL-1β干預(yù)時的軟骨細胞MMP-13含量與濃度為5 ng·mL-1、20 ng·mL-1、25 ng·mL-1的IL-1β干預(yù)后軟骨細胞MMP-13含量比較,差異均無統(tǒng)計學(xué)意義(LSD-t=-2.049,P=0.055; LSD-t=-2.083,P=0.052; LSD-t=0.496,P=0.626); 濃度為10 ng·mL-1、15 ng·mL-1的IL-1β干預(yù)后軟骨細胞MMP-13含量高于未經(jīng)IL-1β干預(yù)時的軟骨細胞MMP-13含量(LSD-t=-3.412,P=0.003; LSD-t=-4.216,P=0.001); 濃度為10 ng·mL-1的IL-1β干預(yù)后軟骨細胞MMP-13含量與濃度為15 ng·mL-1的IL-1β干預(yù)后軟骨細胞MMP-13含量比較,差異無統(tǒng)計學(xué)意義(LSD-t=-0.804,P=0.432)。④軟骨細胞中G蛋白偶聯(lián)信號傳導(dǎo)系統(tǒng)關(guān)鍵調(diào)控因子的蛋白表達。空白血清組、模型組和獨活寄生湯含藥血清組的Gαs、Gαq、Gαo和Gαi蛋白表達量比較,組間差異均有統(tǒng)計學(xué)意義[(0.81±0.09)kDa,(0.31±0.07)kDa,(0.78±0.13)kDa,F=23.669,P=0.001;(0.22±0.04)kDa,(0.14±0.02)kDa,(0.20±0.02)kDa,F=6.500,P=0.031;(0.25±0.02)kDa,(0.12±0.01)kDa,(0.18±0.03)kDa,F=27.214,P=0.001;(0.21±0.02)kDa,(0.26±0.02)kDa,(0.19±0.03)kDa,F=6.882,P=0.028]; 空白血清組Gαs、Gαq、Gαo蛋白表達量高于模型組(LSD-t=6.134,P=0.001; LSD-t=7.370,P=0.000; LSD-t=3.465,P=0.013),Gαi蛋白表達量低于模型組(LSD-t=2.572,P=0.042); 模型組Gαs、Gαq、Gαo蛋白表達量低于獨活寄生湯含藥血清組(LSD-t=5.766,P=0.001; LSD-t=3.401,P=0.014; LSD-t=2.599,P=0.041),Gαi蛋白表達量高于獨活寄生湯含藥血清組(LSD-t=3.609,P=0.011)。結(jié)論:獨活寄生湯含藥血清可以抑制IL-1β誘導(dǎo)的軟骨細胞炎癥反應(yīng),其作用機制可能與G蛋白偶聯(lián)信號傳導(dǎo)系統(tǒng)的調(diào)控有關(guān),但其具體作用靶點有待進一步深入研究。
Abstract:
ABSTRACT Objective:To explore the mechanism of action of Duhuo Jisheng Tang(獨活寄生湯,DHJST)medicated serum in inhibiting inflammatory reaction induced by interleukin-1 beta(IL-1β)in chondrocytes.Methods:Ten 2-month-old male SD rats were randomly divided into normal group and DHJST group.The rats in DHJST group were intragastric administrated with DHJST in dosage of 9.3 g/kg,while the others in normal group were intragastric administrated with the same dose of normal saline,twice per day for 1 consecutive week.After the last intragastric administration,their blood were fetched out from abdominal aorta and were made into DHJST medicated serum and blank serum respectively and the serum were reserved at low temperature for future use.The knee articular cartilage of six 4-week-old SD rats were fetched out and the chondrocytes were isolated and cultured by using enzymatic digestion method.The chondrocytes morphology were observed under optical microscope,and immunohistochemical identification were carried out by using typeⅡcollagenase.The well-growned second-generation chondrocytes were fetched out,and their cytoactive were detected by using MTT method after cultured in medicated serum for 24,36,48 and 72 hours,and the matrix metalloproteinase(MMP)-13 content of chondrocytes were detected by using enzyme-linked immunoadsordent assay(ELISA)after intervention with different concentrations of IL-1β.The second-generation chondrocytes were randomly divided into blank serum group,model group and DHJST medicated serum group.The chondrocytes in blank serum group were cultured in dulbecco modified eagle medium(DMEM)supplemented with 10% blank serum,while the chondrocytes in model group and DHJST medicated serum group were intervened by IL-1β with concentration of 15 ng/ml for 24 hours and then were cultured in DMEM supplemented with 10% blank serum and 10% DHJST medicated serum respectively.The protein expression of key regulating factor of G protein coupled signal transduction systems in chondrocytes were detected by using Western blot method after the chondrocytes were cultured for continuous 48 hours in the 3 groups.Results:The second-generation chondrocytes had plentiful kytoplasm and the refractive extracellular matrix(ECM)could be found around the cells.The cells grew in the form of paving-stone and their cytoplasms presented with tawny positive staining.There was statistical difference in the cytoactive between chondrocytes cultured in medicated serum for 24,36,48 and 72 hours(1.01+/-0.01,1.03+/-0.02,1.07+/-0.01,1.02+/-0.02,F=8.300,P=0.008).The chondrocyte activities were lower at 24 hours compared to at 48 hours after cultivation in medicated serum(t=-4.648,P=0.002).There was no statistical difference in the chondrocyte activities between 24-hour cultivation and 36- and 72-hour cultivation(t=-1.549,P=0.160; t=-0.775,P=0.461).There was statistical difference in the MMP-13 contents between chondrocytes cultured in DMEM supplemented with IL-1β with concentration of 0,5,10,15,20 and 25 ng/ml for 24 hours(0.07+/-0.01,0.08+/-0.01,0.09+/-0.01,0.10+/-0.01,0.08+/-0.01,0.06+/-0.01,F=6.823,P=0.001).There was no statistical difference in the MMP-13 contents of chondrocytes between pre-intervention by IL-1β and post-intervention by IL-1β with concentration of 5,20 and 25 ng/ml respectively(LSD-t=-2.049,P=0.055; LSD-t=-2.083,P=0.052; LSD-t=0.496,P=0.626.The MMP-13 contents of chondrocytes were higher after intervention by IL-1βwith concentration of 10 and 15 ng/ml compared to pre-intervention by IL-1β(LSD-t=-3.412,P=0.003; LSD-t=-4.216,P=0.001).There was no statistical difference in the MMP-13 contents between chondrocytes intervented by IL-1βwith concentration of 10 ng/ml and those intervented by IL-1βwith concentration of 15 ng/ml(LSD-t=-0.804,P=0.432).There was statistical difference in the protein expression of key regulating factor of G protein coupled signal transduction systems including Gαs,Gαq,Gαo and Gαi in chondrocytes between the 3 groups(0.81+/-0.09,0.31+/-0.07,0.78+/-0.13 kDa,F=23.669,P=0.001; 0.22+/-0.04,0.14+/-0.02,0.20+/-0.02 kDa,F=6.500,P=0.031; 0.25+/-0.02,0.12+/-0.01,0.18+/-0.03 kDa,F=27.214,P=0.001; 0.21+/-0.02,0.26+/-0.02,0.19+/-0.03 kDa,F=6.882,P=0.028).The protein expressions of Gαs,Gαq and Gαo were higher and the protein expressions of Gαi were lower in blank serum group compared to model group(LSD-t=6.134,P=0.001; LSD-t=7.370,P=0.000; LSD-t=3.465,P=0.013,LSD-t=2.572,P=0.042).The protein expressions of Gαs,Gαq and Gαo were lower and the protein expressions of Gαi were higher in model group compared to DHJST medicated serum group(LSD-t=5.766,P=0.001; LSD-t=3.401,P=0.014; LSD-t=2.599,P=0.041; LSD-t=3.609,P=0.011).Conclusion:DHJST medicated serum can inhibit inflammatory reaction induced by interleukin-1 beta in chondrocytes.The mechanisms of action may be related to the regulation of G protein coupled signal transduction systems.However,its specific action targets need to be further studied.

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備注/Memo

備注/Memo:
基金項目:國家自然科學(xué)基金項目(81373818) 通訊作者:劉獻祥 E-mail:[email protected]
更新日期/Last Update: 2017-12-29