[1]陈德塔,樊天佑,谢晓亮,等.附子含药血清对脂多糖诱导的软骨细胞凋亡的影响及作用机制研究[J].中医正骨,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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附子含药血清对脂多糖诱导的软骨细胞凋亡的影响及作用机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第34卷
期数:
2022年12期
页码:
1-7,13
栏目:
基础研究
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:
陈德塔1; 樊天佑1; 谢晓亮1; 朱海霞1; 沈雪2; 圣小平1
(1.上海中医药大学附属市中医医院,上海 200071; 2.上海中医药大学附属曙光医院,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
关键词:
骨关节炎;  软骨细胞;  细胞凋亡;  附子;  含药血清;  脂多糖类;  线粒体
Keywords:
osteoarthritis;  chondrocytes;  apoptosis;  aconiti lateralis radix praeparaka;  medicated serum;  lipopolysaccharides;  mitochondria
摘要:
目的:观察附子含药血清对脂多糖诱导的软骨细胞凋亡的影响,并从线粒体功能角度探讨其作用机制。方法:体外分离培养裸鼠膝关节软骨细胞,通过观察细胞形态和甲苯胺蓝染色进行软骨细胞鉴定。通过脂多糖诱导建立软骨细胞退变模型,并采用实时荧光PCR法测定软骨细胞中白细胞介素-6 mRNA、肿瘤坏死因子-α mRNA、基质金属蛋白酶-3 mRNA、基质金属蛋白酶-13 mRNA表达量进行模型鉴定。分别按照0.001 2 mL·g-1、0.002 4 mL·g-1、0.004 8 mL·g-1体质量以制备的附子汤剂和0.9%生理盐水(1 mL)给SD大鼠灌胃,获取低、中、高剂量附子含药血清及空白血清,经CCK-8法检测含药血清细胞毒性,确定2%含药血清为最佳浓度。取培养的第3代软骨细胞,设置6组,附子低、中、高剂量组及空白血清组分别置于加入含1 μg·mL-1脂多糖、2%含药血清、8%FBS的DMEM完全培养基中培养,脂多糖组置于含1 μg·mL-1脂多糖的DMEM完全培养基中培养,对照组在DMEM完全培养基中培养,各组均干预24 h。干预结束后,检测各组软骨细胞中的ATP含量(荧光发光法)、Casepase-3 mRNA表达量(实时荧光PCR法)、Casepase-3蛋白表达量(Western Blot法)及细胞凋亡率(流式细胞术)。结果:①软骨细胞中ATP含量。各组软骨细胞中ATP含量比较,差异有统计学意义(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)。脂多糖组的ATP含量低于对照组和空白血清组(P=0.046,P=0.000); 附子低、中剂量组的ATP含量均高于空白血清组(P=0.018,P=0.013),附子高剂量组与空白血清组ATP含量的差异无统计学意义(P=0.238); 附子低剂量组的ATP含量高于附子高剂量组(P=0.031),附子中剂量组与附子低、高剂量组ATP含量的差异均无统计学意义(P=0.086,P=0.298)。②软骨细胞中Casepase-3 mRNA和Casepase-3蛋白表达量。各组Casepase-3 mRNA表达量比较,差异有统计学意义(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)。脂多糖组的Casepase-3 mRNA表达量高于对照组(P=0.030),脂多糖组与空白血清组Casepase-3 mRNA表达量的差异无统计学意义(P=0.455); 附子低、中、高剂量组的Casepase-3 mRNA表达量均低于空白血清组(P=0.014,P=0.049,P=0.008); 附子低剂量组与附子中、高剂量组Casepase-3 mRNA表达量的组间差异均无统计学意义(P=0.512,P=0.779),附子中、高剂量组Casepase-3 mRNA表达量的差异无统计学意义(P=0.355)。各组Casepase-3蛋白表达量比较,差异有统计学意义(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)。脂多糖组的Casepase-3蛋白表达量高于对照组(P=0.032),脂多糖组与空白血清组Casepase-3蛋白表达量的差异无统计学意义(P=0.455); 附子低、中剂量组与空白血清组Casepase-3蛋白表达量的组间差异均无统计学意义(P=0.089,P=0.165),附子高剂量组的Casepase-3蛋白表达量低于空白血清组(P=0.019); 附子低剂量组与附子中、高剂量组Casepase-3蛋白表达量的组间差异均无统计学意义(P=0.878,P=0.180),附子中、高剂量组Casepase-3蛋白表达量的差异无统计学意义(P=0.999)。③软骨细胞凋亡率。各组软骨细胞凋亡率比较,差异有统计学意义[(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]。脂多糖组的细胞凋亡率高于对照组(P=0.000),脂多糖组与空白血清组细胞凋亡率的差异无统计学意义(P=0.115); 附子低剂量组与空白血清组细胞凋亡率的差异无统计学意义(P=0.105),附子中、高剂量组的细胞凋亡率均低于空白血清组(P=0.003,P=0.000); 附子低剂量组的细胞凋亡率高于附子高剂量组(P=0.030),附子中剂量组与附子低、高剂量组细胞凋亡率的组间差异均无统计学意义(P=0.076,P=0.100)。结论:附子含药血清可以抑制脂多糖诱导的软骨细胞凋亡,这可能与其作用于线粒体依赖性Caspase-3信号通路有关。
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 本文编辑:李晓乐)

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备注/Memo

备注/Memo:
基金项目:上海中医药大学“研究生创新培养”专项科研项目(Y2020040) 通讯作者:圣小平 E-mail:shengxiaop@126.com
更新日期/Last Update: 1900-01-01