Study on the mechanism of Oridonin against pathological damage of tuberculosis

doi:10.19982/j.issn.1000-6621.20220060

Abstract

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

CLC Number:

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

Figures/Tables 4

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

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

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

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

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