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[1]陳德塔,樊天佑,謝曉亮,等.附子含藥血清對脂多糖誘導(dǎo)的軟骨細胞凋亡的影響及作用機制研究[J].中醫(yī)正骨,2022,34(12):1-7,13.
　CHEN Deta,FAN Tianyou,XIE Xiaoliang,et al.Effect and mechanism of Aconiti Lateralis Radix Praeparata medicated serum on apoptosis of chondrocytes induced by lipopolysaccharide[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2022,34(12):1-7,13.
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附子含藥血清對脂多糖誘導(dǎo)的軟骨細胞凋亡的影響及作用機制研究()
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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12期
頁碼:
1-7,13
欄目:
基礎(chǔ)研究
出版日期:
2022-12-02

文章信息/Info

Title:
Effect and mechanism of Aconiti Lateralis Radix Praeparata medicated serum on apoptosis of chondrocytes induced by lipopolysaccharide
作者:
陳德塔1; 樊天佑1; 謝曉亮1; 朱海霞1; 沈雪2; 圣小平1
(1.上海中醫(yī)藥大學(xué)附屬市中醫(yī)醫(yī)院,上海 200071; 2.上海中醫(yī)藥大學(xué)附屬曙光醫(yī)院,上海 201203)
Author(s):
CHEN Deta1; FAN Tianyou1; XIE Xiaoliang1; ZHU Haixia1; SHEN Xue2; SHENG Xiaoping1
1.Traditional Chinese Medicine Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200071,China 2.Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China
關(guān)鍵詞:
骨關(guān)節(jié)炎;  軟骨細胞;  細胞凋亡;  附子;  含藥血清;  脂多糖類;  線粒體
Keywords:
osteoarthritis;  chondrocytes;  apoptosis;  aconiti lateralis radix praeparaka;  medicated serum;  lipopolysaccharides;  mitochondria
摘要:
目的:觀察附子含藥血清對脂多糖誘導(dǎo)的軟骨細胞凋亡的影響,并從線粒體功能角度探討其作用機制。方法:體外分離培養(yǎng)裸鼠膝關(guān)節(jié)軟骨細胞,通過觀察細胞形態(tài)和甲苯胺藍染色進行軟骨細胞鑒定。通過脂多糖誘導(dǎo)建立軟骨細胞退變模型,并采用實時熒光PCR法測定軟骨細胞中白細胞介素-6 mRNA、腫瘤壞死因子-α mRNA、基質(zhì)金屬蛋白酶-3 mRNA、基質(zhì)金屬蛋白酶-13 mRNA表達量進行模型鑒定。分別按照0.001 2 mL·g-1、0.002 4 mL·g-1、0.004 8 mL·g-1體質(zhì)量以制備的附子湯劑和0.9%生理鹽水(1 mL)給SD大鼠灌胃,獲取低、中、高劑量附子含藥血清及空白血清,經(jīng)CCK-8法檢測含藥血清細胞毒性,確定2%含藥血清為最佳濃度。取培養(yǎng)的第3代軟骨細胞,設(shè)置6組,附子低、中、高劑量組及空白血清組分別置于加入含1 μg·mL-1脂多糖、2%含藥血清、8%FBS的DMEM完全培養(yǎng)基中培養(yǎng),脂多糖組置于含1 μg·mL-1脂多糖的DMEM完全培養(yǎng)基中培養(yǎng),對照組在DMEM完全培養(yǎng)基中培養(yǎng),各組均干預(yù)24 h。干預(yù)結(jié)束后,檢測各組軟骨細胞中的ATP含量(熒光發(fā)光法)、Casepase-3 mRNA表達量(實時熒光PCR法)、Casepase-3蛋白表達量(Western Blot法)及細胞凋亡率(流式細胞術(shù))。結(jié)果:①軟骨細胞中ATP含量。各組軟骨細胞中ATP含量比較,差異有統(tǒng)計學(xué)意義(1.294±0.132,1.000±0.000,1.668±0.092,2.633±0.244,2.056±0.110,1.854±0.107,F=99.725,P=0.000)。脂多糖組的ATP含量低于對照組和空白血清組(P=0.046,P=0.000); 附子低、中劑量組的ATP含量均高于空白血清組(P=0.018,P=0.013),附子高劑量組與空白血清組ATP含量的差異無統(tǒng)計學(xué)意義(P=0.238); 附子低劑量組的ATP含量高于附子高劑量組(P=0.031),附子中劑量組與附子低、高劑量組ATP含量的差異均無統(tǒng)計學(xué)意義(P=0.086,P=0.298)。②軟骨細胞中Casepase-3 mRNA和Casepase-3蛋白表達量。各組Casepase-3 mRNA表達量比較,差異有統(tǒng)計學(xué)意義(0.783±0.020,0.996±0.086,0.929±0.083,0.680±0.189,0.739±0.125,0.656±0.044,F=5.003,P=0.010)。脂多糖組的Casepase-3 mRNA表達量高于對照組(P=0.030),脂多糖組與空白血清組Casepase-3 mRNA表達量的差異無統(tǒng)計學(xué)意義(P=0.455); 附子低、中、高劑量組的Casepase-3 mRNA表達量均低于空白血清組(P=0.014,P=0.049,P=0.008); 附子低劑量組與附子中、高劑量組Casepase-3 mRNA表達量的組間差異均無統(tǒng)計學(xué)意義(P=0.512,P=0.779),附子中、高劑量組Casepase-3 mRNA表達量的差異無統(tǒng)計學(xué)意義(P=0.355)。各組Casepase-3蛋白表達量比較,差異有統(tǒng)計學(xué)意義(0.780±0.031,1.000±0.000,0.963±0.055,0.757±0.011,0.671±0.114,0.669±0.034,F=20.213,P=0.000)。脂多糖組的Casepase-3蛋白表達量高于對照組(P=0.032),脂多糖組與空白血清組Casepase-3蛋白表達量的差異無統(tǒng)計學(xué)意義(P=0.455); 附子低、中劑量組與空白血清組Casepase-3蛋白表達量的組間差異均無統(tǒng)計學(xué)意義(P=0.089,P=0.165),附子高劑量組的Casepase-3蛋白表達量低于空白血清組(P=0.019); 附子低劑量組與附子中、高劑量組Casepase-3蛋白表達量的組間差異均無統(tǒng)計學(xué)意義(P=0.878,P=0.180),附子中、高劑量組Casepase-3蛋白表達量的差異無統(tǒng)計學(xué)意義(P=0.999)。③軟骨細胞凋亡率。各組軟骨細胞凋亡率比較,差異有統(tǒng)計學(xué)意義[(6.460±0.806)%,(17.293±1.143)%,(15.413±0.145)%,(13.383±2.290)%,(11.023±1.950)%,(8.920±0.484)%,F=27.103,P=0.000]。脂多糖組的細胞凋亡率高于對照組(P=0.000),脂多糖組與空白血清組細胞凋亡率的差異無統(tǒng)計學(xué)意義(P=0.115); 附子低劑量組與空白血清組細胞凋亡率的差異無統(tǒng)計學(xué)意義(P=0.105),附子中、高劑量組的細胞凋亡率均低于空白血清組(P=0.003,P=0.000); 附子低劑量組的細胞凋亡率高于附子高劑量組(P=0.030),附子中劑量組與附子低、高劑量組細胞凋亡率的組間差異均無統(tǒng)計學(xué)意義(P=0.076,P=0.100)。結(jié)論:附子含藥血清可以抑制脂多糖誘導(dǎo)的軟骨細胞凋亡,這可能與其作用于線粒體依賴性Caspase-3信號通路有關(guān)。
Abstract:
Objective:To observe the effect of Aconiti Lateralis Radix Praeparata(ALRP)medicated serum on the apoptosis of chondrocytes induced by lipopolysaccharide(LPS)and explore the underlying mechanism from the perspective of mitochondrial function.Methods:The knee chondrocytes of nude mice were isolated and cultured in vitro and identified by observation of cell morphology and toluidine blue staining.The degeneration model was induced by LPS in chondrocytes,and the mRNA expression levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-3(MMP-3),and matrix metalloproteinase-13(MMP-13)in chondrocytes were determined by real-time fluorescence-based PCR for model identification.SD rats received ALRP decoction at 0.001 2,0.002 4,and 0.004 8 mL/g and 0.9% normal saline(1 mL)by gavage,and low-,medium-,and high-dose ALRP-medicated serum and blank serum were obtained.The cytotoxicity of ALRP-medicated serum was determined by CCK-8 method,and 2% was the optimal concentration.The chondrocytes of the third generation were cultured and divided into six groups.The cells in the low-,medium-,and high-dose ALRP groups and the blank serum group were cultured in DMEM complete medium containing 1 μg/mL of LPS,2% ALRP-medicated serum,and 8% FBS,and those in the LPS group were cultured in DMEM complete medium containing 1 μg/mL of LPS.The cells in the control group were cultured in DMEM complete medium.After 24 h of intervention,the content of ATP(fluorescence method),mRNA expression of Casepase-3(real-time fluorescence-based PCR),protein expression of Casepase-3(Western blot)and apoptosis rate of chondrocytes(flow cytometry)in each group were determined.Results:①ATP content in chondrocytes.There were significant differences in the content of ATP in chondrocytes of different groups(1.294±0.132,1.000±0.000,1.668±0.092,2.633±0.244,2.056±0.110,1.854±0.107,F=99.725,P=0.000).ATP content in the LPS group was lower than that in the control group and the blank serum group(P=0.046,P=0.000).The content of ATP in the low- and medium-dose ALRP groups was higher than that in the blank serum group(P=0.018,P=0.013).There was no significant difference in the ATP content between the high-dose ALRP group and the blank serum group(P=0.238).The ATP content of the low-dose ALRP group was higher than that of the high-dose ALRP group(P=0.031),and there was no significant difference in the ATP content between the medium-dose ALRP group and the low- and high-dose ALRP groups(P=0.086,P=0.298).②Casepase-3 mRNA and protein expression in chondrocytes.There were significant differences in mRNA expression of Casepase-3 between different groups(0.783±0.020,0.996±0.086,0.929±0.083,0.680±0.189,0.739±0.125,0.656±0.044,F=5.003,P=0.010).The mRNA expression of Casepase-3 in the LPS group was higher than that in the control group(P=0.030),but there was no significant difference between the LPS group and the blank serum group(P=0.455).The mRNA expression of Casepase-3 in the low-,medium-,and high-dose ALRP groups was lower than that in the blank serum group(P=0.014,P=0.049,P=0.008).There was no significant difference in Casepase-3 mRNA expression between the low-dose ALRP group and the medium- and high-dose ALRP groups(P=0.512,P=0.779),and no significant difference in Casepase-3 mRNA expression between the medium-dose ALRP group and the high-dose ALRP group was observed(P=0.355).The differences in Casepase-3 protein expression between different groups were statistically significant(0.780±0.031,1.000±0.000,0.963±0.055,0.757±0.011,0.671±0.114,0.669±0.034,F=20.213,P=0.000).The protein expression of Casepase-3 in the LPS group was higher than that in the control group(P=0.032),but there was no significant difference between the LPS group and the blank serum group(P=0.455).There was no significant difference in Casepase-3 protein expression between the low- and medium-dose ALRP groups and the blank serum group(P=0.089,P=0.165).The expression of Casepase-3 protein in the high-dose ALRP group was lower than that in the blank serum group(P=0.019).There was no significant difference in the expression of Casepase-3 protein between the low-dose ALRP group and the medium- and high-dose ALRP groups(P=0.878,P=0.180).There was no significant difference in Casepase-3 protein expression between the medium-dose ALRP group and the high-dose ALRP group(P=0.999).③Apoptosis rate of chondrocytes.The differences in the apoptosis rate of chondrocytes in different groups were statistically significant(6.460±0.806,17.293±1.143,15.413±0.145,13.383±2.290,11.023±1.950,8.920±0.484%,F=27.103,P=0.000).The apoptosis rate of the LPS group was higher than that of the control group(P=0.000),but there was no significant difference between the LPS group and the blank serum group(P=0.115).There was no significant difference in the apoptosis rate between the low-dose ALRP group and the blank serum group(P=0.105).The apoptosis rates in the medium- and high-dose ALRP groups were lower than that in the blank serum group(P=0.003,P=0.000).The apoptosis rate of the low-dose ALRP group was higher than that of the high-dose ALRP group(P=0.030),and there was no significant difference between the medium-dose ALRP group and the low- and high-dose ALRP groups(P=0.076,P=0.100).Conclusion:The ALRP-medicated serum can inhibit the apoptosis of chondrocytes induced by LPS,which may be attributed to its effect on mitochondria-dependent Caspase-3 signaling pathway.

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(收稿日期:2022-03-13 本文編輯:李曉樂)

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備注/Memo

備注/Memo:
基金項目:上海中醫(yī)藥大學(xué)“研究生創(chuàng)新培養(yǎng)”專項科研項目(Y2020040) 通訊作者:圣小平 E-mail:[email protected]
更新日期/Last Update: 1900-01-01