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[1]王慶敏,沈毅弘,李毅嵩,等.復(fù)方杜仲湯干預(yù)終板軟骨退變作用機制的實驗研究[J].中醫(yī)正骨,2024,36(07):1-9,16.
　WANG Qingmin,SHEN Yihong,LI Yisong,et al.The mechanism of Fufang Duzhong Tang(復(fù)方杜仲湯)against degeneration of cartilage endplate:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(07):1-9,16.
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復(fù)方杜仲湯干預(yù)終板軟骨退變作用機制的實驗研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期數(shù):
2024年07期
頁碼:
1-9,16
欄目:
基礎(chǔ)研究
出版日期:
2024-07-20

文章信息/Info

Title:
The mechanism of Fufang Duzhong Tang(復(fù)方杜仲湯)against degeneration of cartilage endplate:an experimental study
作者:
王慶敏; 沈毅弘; 李毅嵩; 謝強; 鄭慶豐; 沈鴻輝
福建省漳州市中醫(yī)院,福建 漳州 363000
Author(s):
WANG Qingmin; SHEN Yihong; LI Yisong; XIE Qiang; ZHENG Qingfeng; SHEN Honghui
Zhangzhou Traditional Chinese Medical Hospital,Zhangzhou 363000,Fujian,China
關(guān)鍵詞:
軟骨疾病;  終板軟骨;  復(fù)方杜仲湯;  軟骨細(xì)胞;  大鼠;  Sprague-Dawley;  實驗研究
Keywords:
cartilage diseases;  endplate cartilage;  Fufang Duzhong Tang;  chondrocytes;  rats; Sprague-Dawley;  experimental study
摘要:
目的:探討復(fù)方杜仲湯干預(yù)終板軟骨退變的作用機制。方法:取4周齡SD大鼠80只,雌雄各半,隨機分為中藥低劑量組、中藥中劑量組、中藥高劑量組和空白組,中藥低、中、高劑量組大鼠每日灌胃相應(yīng)劑量的復(fù)方杜仲湯藥液,空白組每次用與中藥低劑量組等體積的蒸餾水灌胃,早晚各1次,共灌胃7 d。灌胃干預(yù)結(jié)束后24 h,抽取各組大鼠腹主動脈血,制備相應(yīng)藥物濃度的含藥血清和空白血清。取4周齡SD大鼠60只,雌雄各半,摘取終板軟骨組織,分離、提取終板軟骨細(xì)胞。取對數(shù)生長期的終板軟骨細(xì)胞,分為空白對照組、模型組、空白血清組、低劑量含藥血清組、中劑量含藥血清組和高劑量含藥血清組。除空白對照組外,其他5組細(xì)胞均加入白細(xì)胞介素(interleukin,IL)-1β進行誘導(dǎo)。誘導(dǎo)后,低、中、高劑量含藥血清組和空白血清組分別加入制備的低、中、高劑量的含藥血清和空白血清進行干預(yù)。觀察復(fù)方杜仲湯對終板軟骨細(xì)胞活性、氧化應(yīng)激和炎癥因子水平,以及Kelch樣環(huán)氧氯丙烷相關(guān)蛋白1(kelch-like ECH-associated protein 1,Keap1)-核轉(zhuǎn)錄因子紅系2相關(guān)因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)/抗氧化響應(yīng)元件(antioxidant response element,ARE)信號通路的影響。采用細(xì)胞計數(shù)試劑盒檢測各組終板軟骨細(xì)胞的活性,計算細(xì)胞存活率。采用酶聯(lián)免疫吸附法檢測終板軟骨細(xì)胞中丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、過氧化氫酶(catalase,CAT)、腫瘤壞死因子-α(tumor necrosis factor,TNF-α)、IL-1β、IL-6水平。采用實時定量PCR擴增法檢測終板軟骨細(xì)胞中Nrf2、Keap1、p53、Runt相關(guān)轉(zhuǎn)錄因子2(runt-related transcription factor 2,Runx2)、基質(zhì)金屬蛋白酶13(matrix metalloproteinase 13,MMP13)和Y染色體性別決定區(qū)-盒轉(zhuǎn)錄因子9(sex-determing region of Y chromosome-box transcription factor 9,Sox9)的mRNA相對表達量。采用蛋白質(zhì)印跡法檢測終板軟骨細(xì)胞中Nrf2、Keap1、P53、Runx2、MMP13和Sox9蛋白相對表達量。結(jié)果:①復(fù)方杜仲湯對終板軟骨細(xì)胞活性影響的檢測結(jié)果。模型組、空白血清組、低劑量含藥血清組、中劑量含藥血清組和高劑量含藥血清組終板軟骨細(xì)胞存活率均低于空白對照組。低、中、高劑量含藥血清組細(xì)胞存活率均高于模型組和空白血清組。中、高劑量含藥血清組細(xì)胞存活率均高于低劑量含藥血清組。高劑量含藥血清組細(xì)胞存活率高于中劑量含藥血清組。②復(fù)方杜仲湯對終板軟骨細(xì)胞氧化應(yīng)激和炎癥因子水平影響的檢測結(jié)果。模型組、空白血清組、低劑量含藥血清組、中劑量含藥血清組和高劑量含藥血清組終板軟骨細(xì)胞中MDA、TNF-α、IL-1β和IL-6水平均高于空白對照組,SOD、CAT水平均低于空白對照組。低、中、高劑量含藥血清組終板軟骨細(xì)胞中MDA、TNF-α、IL-1β、IL-6水平均低于模型組,SOD、CAT水平均高于模型組和空白血清組。中、高劑量含藥血清組終板軟骨細(xì)胞中MDA、TNF-α、IL-1β和IL-6水平均低于低劑量含藥血清組,SOD、CAT水平均高于低劑量含藥血清組。高劑量含藥血清組MDA、TNF-α、IL-1β和IL-6水平均低于中劑量含藥血清組,SOD、CAT水平均高于中劑量含藥血清組。③復(fù)方杜仲湯對終板軟骨細(xì)胞中Keap1-Nrf2/ARE信號通路影響的檢測結(jié)果。模型組、空白血清組、低劑量含藥血清組、中劑量含藥血清組和高劑量含藥血清組終板軟骨細(xì)胞中Keap1、p53、Runx2、MMP13的mRNA相對表達量和蛋白相對表達量均高于空白對照組,Nrf2、Sox9的mRNA相對表達量和蛋白相對表達量均低于空白對照組。低、中、高劑量含藥血清組終板軟骨細(xì)胞中Keap1、p53、Runx2、MMP13的mRNA相對表達量和蛋白相對表達量均低于模型組和空白血清組,Nrf2、Sox9的mRNA相對表達量和蛋白相對表達量均高于模型組和空白血清組。中、高劑量含藥血清組終板軟骨細(xì)胞中Keap1、p53、Runx2、MMP13的mRNA相對表達量和蛋白相對表達量均低于低劑量含藥血清組,Nrf2、Sox9的mRNA相對表達量和蛋白相對表達量均高于低劑量含藥血清組。高劑量含藥血清組終板軟骨細(xì)胞中Keap1、p53、Runx2、MMP13的mRNA相對表達量和蛋白相對表達量均低于中劑量含藥血清組,Nrf2、Sox9的mRNA相對表達量和蛋白相對表達量均高于中劑量含藥血清組。結(jié)論:復(fù)方杜仲湯可能通過調(diào)節(jié)Keap1-Nrf2/ARE信號通路相關(guān)基因的表達,提高終板軟骨細(xì)胞的活性,降低細(xì)胞內(nèi)氧化應(yīng)激和炎癥因子水平,從而起到干預(yù)終板軟骨退變的作用,且其干預(yù)效果呈劑量依賴性。
Abstract:
Objective:To explore the mechanism of Fufang Duzhong Tang(復(fù)方杜仲湯,FFDZT)against cartilage endplate(CEP)degeneration.Methods:Eighty 4-week-old Sprague-Dawley(SD)rats,half in males and females,were selected and randomized into low-dose FFDZT(L-FFDZT)group,medium-dose FFDZT(M-FFDZT)group,high-dose FFDZT(H-FFDZT)group,and blank group.The rats in L-FFDZT group,M-FFDZT group,and H-FFDZT group were intragastric administrated with FFDZT in daily dosages of 20.92,41.84,83.68 mL/kg,respectively,while the ones in blank group with an equal volume of distilled water as that of L-FFDZT group,once in the morning and evening,respectively,for consecutive 7 days.On hour 24 after the end of drug intervention,the blood was drawn from the abdominal aorta of rats in each group for making blank serum and medicated serum with the corresponding concentrations.Additionally,60 4-week-old SD rats,half in males and females,were selected for harvesting the CEP tissues,and then isolating and extracting the CEP chondrocytes.The CEP chondrocytes in the logarithmic growth phase were selected and divided into the blank control group,model group,blank serum group,low-,medium-,and high-dose medicated serum group.All the CEP chondrocytes but the ones in blank control group were intervened by interleukin(IL)-1β for inducing degeneration.After successful inducing,the degenerated CEP chondrocytes in blank serum group,low-,medium-,and high-dose medicated serum group were further intervened with the prepared blank serum and medicated serum in their corresponding concentration,respectively.After the end of intervention,the effects of FFDZT on the viability of CEP chondrocytes,le-vels of oxidative stress(OS)and inflammatory factors,and kelch-like ECH-associated protein 1(Keap1)-nuclear factor-erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway were observed.Moreover,the viability of CEP chondrocytes in each group was detected by using the cell counting kit-8(CCK8)assay,and the chondrocyte survival rate was calculated,meanwhile,the levels of malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),tumor necrosis factor-α(TNF-α),IL-1β,and IL-6 in CEP chondrocytes were detected by using enzyme-linked immunosorbent assay(ELISA),and the relative mRNA and protein expression levels of Nrf2,Keap1,p53,runt-related transcription factor 2(Runx2),matrix metalloproteinase 13(MMP13),and sex-determining region of Y chromosome-box transcription factor 9(Sox9)in CEP chondrocytes were detected by using real-time quantitative PCR(RT-qPCR)and Western blotting,respectively.Results:①The survival rate of CEP chondrocytes was lower in model group,blank serum group,low-,medium-,and high-dose medicated serum group compared to blank control group,and it was lower in model group and blank serum group compared to low-,medium-,and high-dose medicated serum group,was lower in low-dose medicated serum group compared to medium-,and high-dose medicated serum group,and was lower in medium-dose medicated serum group compared to high-dose medicated serum group.②The levels of MDA,TNF-α,IL-1β,and IL-6 in CEP chondrocytes were higher,while the levels of SOD and CAT were lower in model group,blank serum group,low-,medium-,and high-dose medicated serum group compared to blank control group.The levels of MDA,TNF-α,IL-1β,and IL-6 in CEP chondrocytes were lower in low-,medium-,and high-dose medicated serum group compared to model group,while the levels of SOD and CAT were higher in low-,medium-,and high-dose medicated serum group compared to model group and blank serum group.The levels of MDA,TNF-α,IL-1β,and IL-6 in CEP chondrocytes were lower,while the levels of SOD and CAT were higher in medium- and high-dose medicated serum group compared to low-dose medicated serum group.The levels of MDA,TNF-α,IL-1β,and IL-6 in CEP chondrocytes were lower,while the levels of SOD and CAT were higher in high-dose medicated serum group compared to medium-dose medicated serum group.③The relative mRNA and protein expression levels of Keap1,p53,Runx2,and MMP13 in CEP chondrocytes were higher,while the relative mRNA and protein expression levels of Nrf2 and Sox9 were lower in model group,blank serum group,low-,medium-,and high-dose medicated serum group compared to blank control group.The relative mRNA and protein expression levels of Keap1,p53,Runx2,and MMP13 in CEP chondrocytes were lower,while the relative mRNA and protein expression levels of Nrf2 and Sox9 were higher in low-,medium-,and high-dose medicated serum group compared to model group and blank serum group.The relative mRNA and protein expression levels of Keap1,p53,Runx2,and MMP13 in CEP chondrocytes were lower,while the relative mRNA and protein expression levels of Nrf2 and Sox9 were higher in medium- and high-dose medicated serum group compared to low-dose medicated serum group.The relative mRNA and protein expression levels of Keap1,p53,Runx2,and MMP13 in CEP chondrocytes were lower,while the relative mRNA and protein expression levels of Nrf2 and Sox9 were higher in high-dose medicated serum group compared to medium-dose medicated serum group.Conclusion:FFDZT may enhance the viability of CEP chondrocytes and reduce the levels of intracellular OS and inflammatory factors by regulating the expression of Keap1-Nrf2/ARE signaling pathway-related genes.Therefore,it can intervene the degeneration of CEP,with a dose-dependence intervention effect.

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備注/Memo

備注/Memo:
基金項目:福建省衛(wèi)健委科技計劃項目(2021lzyjc25); 福建省中醫(yī)學(xué)術(shù)流派傳承工作室建設(shè)項目(閩衛(wèi)中醫(yī)函〔2019〕129號)
通訊作者:沈毅弘 E-mail:[email protected]
更新日期/Last Update: 1900-01-01