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[1]許麗梅,李慧,許云騰,等.基于NF-κB信號通路探討獨活寄生湯抑制脂多糖誘導的軟骨細胞炎癥反應的作用機制[J].中醫(yī)正骨,2019,31(07):9-14.
　XU Limei,LI Hui,XU Yunteng,et al.An experimental study of mechanism of action of Duhuo Jisheng Tang(獨活寄生湯)in inhibiting inflammatory reaction induced by lipopolysaccharide in chondrocytes based on NF-κB signaling pathway[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2019,31(07):9-14.
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基于NF-κB信號通路探討獨活寄生湯抑制脂多糖誘導的軟骨細胞炎癥反應的作用機制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第31卷
期數(shù):
2019年07期
頁碼:
9-14
欄目:
基礎研究
出版日期:
2019-07-20

文章信息/Info

Title:
An experimental study of mechanism of action of Duhuo Jisheng Tang(獨活寄生湯)in inhibiting inflammatory reaction induced by lipopolysaccharide in chondrocytes based on NF-κB signaling pathway
作者:
許麗梅1; 李慧2; 許云騰1; 王圣杰2; 曾建偉1; 王麗麗1; 葉蕻芝3; 李西海1
(1.福建中醫(yī)藥大學中西醫(yī)結合研究院,福建 福州 350122; 2.福建中醫(yī)藥大學藥學院,福建 福州 350122; 3.福建省中西醫(yī)結合老年性疾病重點實驗室,福建 福州 350122)
Author(s):
XU Limei1; LI Hui2; XU Yunteng1; WANG Shengjie2; ZENG Jianwei1; WANG Lili1; YE Hongzhi3; LI Xihai1
1.Academy of Integrated Medicine affiliated to Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 2.Pharmaceutical college of Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 3.Fujian Key Laboratory of Integrated Medicine on Geriatrics,Fuzhou 350122,Fujian,China
關鍵詞:
骨關節(jié)炎;  軟骨細胞;  獨活寄生湯;  炎癥反應;  脂多糖類;  NF-κB
Keywords:
osteoarthritis;  chondrocytes;  Duhuo Jisheng Tang;  inflammatory response;  lipopolysaccharides;  NF-kappa B
摘要:
目的:基于NF-κB信號通路探討獨活寄生湯抑制脂多糖誘導的軟骨細胞炎癥反應的作用機制。方法:取4周齡SPF級SD大鼠雙側膝關節(jié)軟骨,分離軟骨細胞進行體外培養(yǎng)。取培養(yǎng)的第2代軟骨細胞,隨機分為5組。空白組中加入含10%FBS的DMEM培養(yǎng)基,模型組加入含10 ng·mL-1脂多糖的10%FBS DMEM培養(yǎng)基,獨活寄生湯低、中、高濃度組加入含脂多糖(10 ng·mL-1)和獨活寄生湯(濃度分別為100 μg·mL-1、200 μg·mL-1、300 μg·mL-1)的10%FBS DMEM培養(yǎng)基。干預8 h后,采用ELISA法測定各組細胞培養(yǎng)液上清液中的基質金屬蛋白酶(matrix metalloproteinase,MMP)-9含量,確定獨活寄生湯最佳干預濃度。隨后取培養(yǎng)的第2代軟骨細胞,隨機分為4組。空白組不進行干預,模型組加入10 ng·mL-1脂多糖,獨活寄生湯組加入10 ng·mL-1脂多糖和最佳濃度的獨活寄生湯,抑制劑組加入10 ng·mL-1脂多糖和10 μmol吡咯烷二硫代甲酸銨鹽。干預8 h后,采用熒光定量PCR法檢測軟骨細胞中NF-κB基因含量、用免疫熒光檢測法觀察軟骨細胞中NF-κB蛋白核轉位情況。結果:①獨活寄生湯最佳干預濃度測定結果。空白組、模型組、獨活寄生湯低濃度組、獨活寄生湯中濃度組、獨活寄生湯高濃度組的MMP-9含量比較,差異有統(tǒng)計學意義[(0.125±0.012)ng·mL-1,(0.154±0.008)ng·mL-1,(0.148±0.012)ng·mL-1,(0.148±0.010)ng·mL-1,(0.134±0.002)ng·mL-1,χ2=10.034,P=0.040]。模型組的MMP-9含量高于空白組和獨活寄生湯高濃度組(P=0.002,P=0.006); 空白組與獨活寄生湯高濃度組比較,差異無統(tǒng)計學意義(P=0.573); 獨活寄生湯低、中濃度組MMP-9含量與模型組比較,差異均無統(tǒng)計學意義(P=0.483,P=0.356)。上述測定結果顯示獨活寄生湯最佳干預濃度為300 μg·mL-1。②軟骨細胞中NF-κB基因含量。空白組、模型組、獨活寄生湯組、抑制劑組的NF-κB基因含量比較,差異有統(tǒng)計學意義[1.013±0.015,1.848±0.216,1.454±0.250,1.289±0.332,F=8.859,P=0.002]。模型組的NF-κB基因含量高于空白組、獨活寄生湯組和抑制劑組(P=0.000,P=0.035,P=0.006); 獨活寄生湯組的NF-κB基因含量高于空白組(P=0.021); 獨活寄生湯組的NF-κB基因含量與抑制劑組比較,差異無統(tǒng)計學意義(P=0.341)。③軟骨細胞中NF-κB蛋白核轉位情況。各組軟骨細胞核均呈藍色; 軟骨細胞NF-κB蛋白染色明顯,呈綠色; 模型組NF-κB蛋白核轉位最為明顯,空白組、抑制劑組NF-κB蛋白核轉位相對較少,獨活寄生湯組NF-κB蛋白核轉位情況介于空白組與模型組之間。結論:獨活寄生湯可通過抑制NF-κB蛋白核轉位,減輕脂多糖誘導的軟骨細胞炎癥反應,從而延緩軟骨退變。
Abstract:
Objective:To explore the mechanism of action of Duhuo Jisheng Tang(獨活寄生湯,DHJST)in inhibiting inflammatory reaction induced by lipopolysaccharide(LPS)in chondrocytes based on NF-κB signaling pathway.Methods:The 4-week-old SPF-grade SD rats were executed and their articular cartilages were fetched out from both knees,and the chondrocytes were extracted from the knee cartilage tissues and were cultured in vitro.The second-generation chondrocytes were divided into 5 groups,and were cultured in DMEM added with 10% FBS(blank group),10% FBS and LPS(10 ng/mL)(model group),10% FBS,LPS(10 ng/mL)and DHJST(100 μg/mL)(DHJST low-concentration group),10% FBS,LPS(10 ng/mL)and DHJST(200 μg/mL)(DHJST middle-concentration group)and 10% FBS,LPS(10 ng/mL)and DHJST(300 μg/mL)(DHJST high-concentration group)respectively.The content of matrix metalloproteinase(MMP)-9 in supernatant of cell culture fluid of each group were measured by using ELISA method after 8-hour intervention to determine the optimal intervention concentration of DHJST.Then the second-generation chondrocytes were randomly divided into blank group,model group,DHJST group and inhibitor group.The chondrocytes in blank group were not intervened and the chondrocytes in the other three groups were cultured in DMEM added with LPS(10 ng/mL),DMEM added with LPS(10 ng/mL)and DHJST with optimal concentration and DMEM added with LPS(10 ng/mL)and PDTC(10 μmol)respectively.After 8-hour intervention,the gene content of NF-κB in chondrocytes were detected by using fluorescent quantitative PCR method,and nuclear translocation of NF-κB protein in chondrocytes were detected by using immunofluorescence assay.Results:There was statistical difference in content of MMP-9 between blank group,model group,DHJST low-,middle- and high-concentration group(0.125+/-0.012,0.154+/-0.008,0.148+/-0.012,0.148+/-0.010,0.134+/-0.002 ng/mL,χ2=10.034,P=0.040).The content of MMP-9 was higher in model group compared to blank group and DHJST high-concentration group(P=0.002,P=0.006),and there was no statistical difference in content of MMP-9 between blank group and DHJST high-concentration group(P=0.573).There was no statistical difference in content of MMP-9 between DHJST low-concentration group and model group and between DHJST middle-concentration group and model group(P=0.483,P=0.356).Above results showed that the optimal intervention concentration of DHJST was 300 μg/mL.There was statistical difference in gene content of NF-κB between blank group,model group,DHJST group and inhibitor group(1.013+/-0.015,1.848+/-0.216,1.454+/-0.250,1.289+/-0.332,F=8.859,P=0.002).The gene content of NF-κB was higher in model group compared to blank group,DHJST group and inhibitor group,and was higher in DHJST group compared to blank group(P=0.000,P=0.035,P=0.006; P=0.021).There was no statistical difference in gene content of NF-κB between DHJST group and inhibitor group(P=0.341).The cytoblast of chondrocytes presented with blue staining in each group.The chondrocytes presented with obvious green staining of NF-κB protein.The most obvious nuclear translocations of NF-κB protein were found in model group,and relatively less nuclear translocations of NF-κB protein were found in blank group and inhibitor group,and the nuclear translocation of NF-κB protein in DHJST group was between blank group and model group.Conclusion:DHJST can alleviate inflammatory reaction induced by LPS in chondrocytes through inhibiting nuclear translocation of NF-κB protein,which may be the mechanisms of action for delaying articular cartilage degeneration.

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備注/Memo

備注/Memo:
基金項目:國家自然科學基金項目(81573998); 中國博士后科學基金第60批面上資助項目(2016M600625); 中國博士后科學基金第10批特別資助項目(2017T100591) 通訊作者:李西海 E-mail:[email protected](收稿日期:2019-06-05 本文編輯:李曉樂)
更新日期/Last Update: 2019-07-20