冬凌草甲素抗结核病理损伤的作用机制研究

doi:10.19982/j.issn.1000-6621.20220060

摘要/Abstract

摘要：

目的: 通过冬凌草甲素对结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染巨噬细胞NOD样受体家族蛋白3(NOD-like receptor protein 3,NLRP3)炎症小体及内质网应激的影响,探讨冬凌草甲素抗结核病理损伤的作用机制。方法: 利用MTB标准株H37Ra建立小鼠巨噬细胞Raw264.7感染模型,设置空白对照组、MTB感染模型组、冬凌草甲素作用不同浓度(0.5、1.0、2.0、4.0μmol/L)及作用不同时间点(6、12、24h)干预组。应用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测冬凌草甲素的可用药物浓度。应用蛋白免疫印迹法检测NLRP3、硫氧还蛋白互作蛋白(thioredoxin-interacting protein,TXNIP)表达情况;检测内质网应激相关蛋白免疫球蛋白结合蛋白(Bip)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、真核生物翻译起始因子2α(peIF2α)、磷酸化肌醇需要酶 1α(pIRE1α)、肌醇需要酶1α(IRE1α)的表达情况;检测内质网应激下游核因子κB/磷酸化应激活化蛋白激酶/p38(NF-κB/pJNK/pp38)通路的变化,并采用Image J软件做蛋白定量分析。结果: 冬凌草甲素在4.0μmol/L浓度以下细胞生存率在90%左右,对细胞毒性较小。在不同时间点(12、24h),与MTB感染模型组相比,冬凌草甲素可明显降低NLRP3蛋白表达(4.35±0.13 vs. 5.95±0.15;1.90±0.05 vs. 3.93±0.09),明显降低TXNIP蛋白表达(1.14±0.05 vs. 1.73±0.04;0.78±0.05 vs. 1.33±0.02),差异均有统计学意义(F值分别为508.308和166.278,P值均为0.000)。在不同时间点(6、12h),与MTB感染模型组相比,冬凌草甲素可明显降低Bip蛋白表达(1.85±0.07 vs. 2.27±0.07;0.97±0.03 vs. 2.28±0.17),明显降低peIF2α蛋白表达(1.75±0.42 vs. 1.75±0.03;1.31±0.04 vs. 2.45±0.17),明显降低IRE1α蛋白表达(10.48±0.40 vs. 14.19±0.45;6.15±0.15 vs. 15.76±1.27),明显降低pp65蛋白表达(0.69±0.01 vs. 1.07±0.03;0.28±0.01 vs. 0.39±0.02),差异均有统计学意义(F值分别为149.510、10.489、10.294、288.194,P值均<0.01)。结论: MTB感染小鼠巨噬细胞Raw264.7模型中,冬凌草甲素可通过调控内质网应激,抑制NLRP3炎症小体活化,发挥抗结核病理损伤的作用。

关键词: 分枝杆菌,结核, 巨噬细胞, 冬凌草属, 炎症介导素类, 内质网

Abstract:

Objective: To explore the mechanism of Oridonin against pathological damage of tuberculosis through its effect on the NOD-like receptor protein 3 (NLRP3) inflammasomes and endoplasmic reticulum stress in Mycobacterium tuberculosis (MTB)-infected macrophages. Methods: H37Ra strain was used to establish macrophage infection model, meanwhile, blank control group, MTB infection model group and Oridonin intervention groups with different concentrations (0.5, 1.0, 2.0, 4.0 μmol/L) and different time points (6, 12, 24 h) were also set up. Methyl thiazolyl tetrazolium (MTT) method was used to detect the safe dosage range of Oridonin. Western blot was used to detect the expression level of NLRP3 and thioredoxin-interacting protein (TXNIP) protein, immunoglobulin binding protein (Bip), CCAAT/enhancer binding protein homologous protein (CHOP), phosphorylated eukaryotic translation initiation factor 2α (peIF2α) and phosphorylated inositol Requires protein expression of enzyme 1α (pIRE1α) which related to endoplasmic reticulum stress. Western blot was also used to detect the changes of NF-κB/pJNK/pp38 pathway located on downstream of endoplasmic reticulum stress. Protein quantitative analysis was performed with Image J software. Results: The survival rate of infected macrophage treated with oridonin below 4.0 μmol/L was about 90%, indicating minor toxic to cells. Western blot showed that compared with the model group at different time points (12, 24 h), Oridonin could significantly reduce expression levels of NLRP3 protein (4.35±0.13 vs. 5.95±0.15; 1.90±0.05 vs. 3.93±0.09) and TXNIP protein (1.14±0.05 vs. 1.73±0.04; 0.78±0.05 vs. 1.33±0.02), the differences were statistically significant (F=508.308 and 166.278; all P=0.000). Compared with the model group at different time points (6 h, 12 h), Oridonin could significantly reduce expression levels of Bip protein (1.85±0.07 vs. 2.27±0.07; 0.97±0.03 vs. 2.28±0.17), peIF2α protein (1.75±0.42 vs. 1.75±0.03; 1.31±0.04 vs. 2.45±0.17), IRE1α protein (10.48±0.40 vs. 14.19±0.45; 6.15±0.15 vs. 15.76±1.27), pp65 protein (0.69±0.01 vs. 1.07±0.03; 0.28±0.01 vs. 0.39±0.02), the differences were statistically significant (F=149.510, 10.489, 10.294, and 288.194, respectively; all P<0.01). Conclusion: Oridonin could regulate endoplasmic reticulum stress to inhibit the activation of NLRP3 inflammasomes, thus exert the anti-pathological damage effect in MTB-infected macrophages.

Key words: Mycobacterium tuberculosis, Macrophages, Rabdosia, Inflammation mediators, Endoplasmic reticulum

中图分类号:

李银虹, 刘芳琳, 鹿振辉, 姜昕. 冬凌草甲素抗结核病理损伤的作用机制研究[J]. 中国防痨杂志, 2022, 44(8): 849-854. doi: 10.19982/j.issn.1000-6621.20220060

Li Yinhong, Liu Fanglin, Lu Zhenhui, Jiang Xin. Study on the mechanism of Oridonin against pathological damage of tuberculosis[J]. Chinese Journal of Antituberculosis, 2022, 44(8): 849-854. doi: 10.19982/j.issn.1000-6621.20220060

图/表 4

表1

不同浓度冬凌草甲素作用下Raw264.7细胞的生存情况

冬凌草甲素 (μmol/L)	24h细胞生存率 (%, $x ¯$ ±s)	48h细胞生存率 (%, $x ¯$ ±s)	72h细胞生存率 (%, $x ¯$ ±s)
空白组	100.0±0.00	100.00±0.00	100.00±0.00
0.5	93.43±0.78	100.70±3.60	90.75±0.64
1.0	94.13±0.95	100.80±1.33	94.38±0.64
2.0	94.03±0.30	100.50±1.32	98.18±0.61
4.0	91.76±1.40	104.10±1.31	101.60±0.61
8.0	83.88±1.63	91.86±2.09	86.16±0.25

表1

表2

冬凌草甲素作用MTB感染的Raw264.7细胞不同时间对NLRP3炎症小体活化的影响

组别	NLRP3蛋白相对灰度值( $x ¯$ ±s)	TXNIP蛋白相对灰度值( $x ¯$ ±s)
空白组	1.00±0.01	1.00±0.04
模型组(6h)	8.37±0.43^a	1.62±0.07^a
模型组(12h)	5.95±0.15^a	1.73±0.04^a
模型组(24h)	3.93±0.85^a	1.33±0.02^a
冬凌草甲素(6h)	5.51±0.07^b	1.05±0.01^b
冬凌草甲素(12h)	4.35±0.13^c	1.14±0.05^c
冬凌草甲素(24h)	1.90±0.05^d	0.78±0.05^d
F值	508.308	166.278
P值	0.000	0.000

表2

表3

冬凌草甲素作用MTB感染的Raw264.7细胞不同时间对内质网应激的影响

组别	Bip蛋白相对灰度值( $x ¯$ ±s)	CHOP蛋白相对灰度值( $x ¯$ ±s)	peIF2α蛋白相对灰度值( $x ¯$ ±s)	IRE1α蛋白相对灰度值( $x ¯$ ±s)	pIRE1α蛋白相对灰度值( $x ¯$ ±s)
空白组	1.02±0.09	1.02±0.12	0.99±0.06	1.07±0.07	0.94±0.07
模型组(6h)	2.27±0.07^a	1.93±0.08^a	1.75±0.03^a	14.19±0.45^a	5.68±0.19^a
模型组(12h)	2.28±0.17^a	3.86±0.32^a	2.45±0.17^a	15.76±1.27^a	7.29±0.41^a
模型组(24h)	3.68±0.07^a	13.02±0.41^a	2.41±0.15^a	15.39±0.88^a	9.45±0.36^a
冬凌草甲素(6h)	1.85±0.07^b	1.03±0.01^b	1.75±0.42^b	10.48±0.40^b	5.22±0.27
冬凌草甲素(12h)	0.97±0.03^c	3.49±0.09^c	1.31±0.04^c	6.15±0.15^c	4.10±0.08^c
冬凌草甲素(24h)	2.11±0.17^d	6.44±0.24^d	0.75±0.03^d	10.47±0.36^d	7.23±0.13^d
F值	149.510	146.073	10.489	10.294	53.538
P值	0.000	0.000	0.002	0.002	0.000

表3

表4

冬凌草甲素作用MTB感染的Raw264.7细胞不同时间对内质网应激IRE1α通路下游相关蛋白的影响

组别	pp65蛋白相对灰度值( $x ¯$ ±s)	pJNK蛋白相对灰度值( $x ¯$ ±s)	pp38蛋白相对灰度值( $x ¯$ ±s)
空白组	1.00±0.06	1.01±0.12	0.98±0.08
模型组(6h)	1.07±0.03	666.00±31.32^a	1.63±0.09^a
模型组(12h)	0.39±0.02^a	471.80±29.38^a	1.53±0.11^a
模型组(24h)	0.89±0.06	334.70±25.68^a	593.10±52.74^a
冬凌草甲素(6h)	0.69±0.01^b	296.70±10.14^b	6.82±0.27^b
冬凌草甲素(12h)	0.28±0.01^c	46.05±0.86^c	1.17±0.08^c
冬凌草甲素(24h)	0.81±0.00	38.74±1.12^d	244.4±4.72^d
F值	288.194	287.059	285.140
P值	0.000	0.000	0.000

表4

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