Study of autophagy induced by myricetin in MTB infected macrophages through PI3K/Akt/mTOR signaling pathway

doi:10.3969/j.issn.1000-6621.2020.02.005

Abstract

Abstract:

Objective To explore the anti-tuberculosis mechanism of myricetin by studying the autophagy of MTB-infected macrophages induced by myricetin through PI3K/Akt/mTOR signaling pathway. Methods CCK8 method was used to detect the effect of myricetin on cell proliferation and determine the safe dosage range. Murine Raw 264.7 macrophages infected with H37Ra strain was used as the model group, meanwhile we set a blank group and a myricetin group. The cells were infected with MTB H37Ra (Multiplicity of infection=10). After 4 h of co-incubation, cells were washed three times with PBS to discard the MTB that did not enter the cells. In order to determine the optimal concentration of myricetin which promoted autophagy, the protein levels of LC3Ⅱ and p62 were detected by western blot in MTB-infected macrophages treated by different concentrations (12.5, 25, 50, 100 μmol/L) of myricetin for 24 h. The intracellular bacterial load of macrophages after treated with myricetin for 72 h were checked using colony-forming unit (CFU) method. Phosphorylations of Akt and mTOR in PI3K/Akt/mTOR signaling pathway were tested with western blot method for those infected macrophages treated with myricetin for 30, 60 and 180 min. Protein quantitative analysis was performed with Image J software. The results were analyzed using GraphPad Prism 7.0, one-way analysis of variance (ANOVA) was used for pairwise comparison of data, and P<0.05 was considered statistically significant. Results The survival rate of infected macrophages treated with myricetin below 100 μmol/L was about 90%, indicating minor toxic to cells. Western blot showed that compared with model group (0.52±0.01), LC3 Ⅱ expression (0.59±0.02, 0.65±0.01, 0.71±0.01, 0.83±0.01) were changed significantly with different concentrations of myricetin (12.5, 25, 50, 100 μmol/L). Differences were statistically significant (t=2.97,P=0.04,t=7.91,P=0.00,t=9.77,P=0.00,t=16.37,P=0.00); Comparing with the model group (0.86±0.02), different concentrations of myricetin treatment also inhibited the expression of p62 (0.72±0.01, 0.85±0.00, 0.60±0.02, 0.58±0.01). Differences were statistically significant (t=6.50, P=0.00,t=9.53, P=0.00,t=12.01, P=0.00) for concentrations of 12.5, 50, 100 μmol/L respectively, while the group treated with 25 μmol/L (t=0.81,P=0.46) was not statistically significant. The optimal drug concentration of myricetin to promote autophagy was 100 μmol/L. The inhibition rate of intracellular MTB was 21.02% when infected macrophages were treated with myricetin for 72 h. The phosphorylation of Akt in PI3K/Akt/mTOR pathway (1.23±0.01, 1.52±0.01, 0.74±0.02) was significantly increased in the model group at 30, 60, and 180 min after MTB infection, while the phosphorylation of Akt was significantly inhibited by myricetin at the same time (0.99±0.01, 0.96±0.01, 0.43±0.01), with statistically significant differences (t=27.60, P=0.00, t=30.06, P=0.00,t=18.60, P=0.00). However, the protein level of phosphorylated mTOR (p-mTOR) in the model group only increased significantly after MTB infection for 180 min (0.57±0.00), and myricetin also inhibited the phosphorylation of mTOR protein for 180 min (0.46±0.01), with a statistically significant difference (t=21.60, P=0.00). Conclusion Myricetin inhibited the PI3K/Akt/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, thus inducing autophagy in MTB-infected macrophages to kill intracellular MTB.

Key words: Mycobacterium tuberculosis, Macrophages, Autophagy, PI3K/Akt/mTOR, Myricetin

SUN Jin-xia,ZHANG Qing-wen,LI Yin-hong,JIANG Xin. Study of autophagy induced by myricetin in MTB infected macrophages through PI3K/Akt/mTOR signaling pathway[J]. Chinese Journal of Antituberculosis, 2020, 42(2): 101-107. doi: 10.3969/j.issn.1000-6621.2020.02.005

Figures/Tables 6

杨梅素 (μmol/L)	24h细胞存活率 (%, $x ˉ$ ±s)	t值^a	P值^a	48h细胞存活率 (%, $x ˉ$ ±s)	t值^b	P值^b	72h细胞存活率 (%, $x ˉ$ ±s)	t值^c	P值^c
空白组	99.35±0.76			101.90±1.28			94.50±2.85
12.5	96.99±3.45	0.67	0.54	92.13±1.82	5.34	0.01	89.75±3.36	1.08	0.34
25	90.68±0.44	9.95	0.00	92.07±2.00	4.13	0.02	89.54±1.49	1.54	0.20
50	88.39±1.26	7.45	0.00	91.57±1.53	5.18	0.01	91.24±0.96	1.09	0.34
100	92.40±0.68	6.83	0.00	93.82±0.47	5.93	0.00	86.48±2.26	2.21	0.09
200	89.03±0.86	9.02	0.00	92.13±1.82	4.38	0.01	79.65±1.36	4.71	0.01

组别	LC3Ⅱ蛋白相对灰度值 ( $x ˉ$ ±s)	t值	P值	p62蛋白相对灰度值 ( $x ˉ$ ±s)	t值	P值
空白组	0.36±0.01			0.51±0.02
模型组	0.52±0.01	8.85^a	0.00^a	0.86±0.02	10.98^c	0.00^c
杨梅素12.5μmol/L组	0.59±0.02	2.97^b	0.04^b	0.72±0.01	6.50^d	0.00^d
杨梅素25μmol/L组	0.65±0.01	7.91^b	0.00^b	0.85±0.00	0.81^d	0.46^d
杨梅素50μmol/L组	0.71±0.01	9.77^b	0.00^b	0.60±0.02	9.53^d	0.00^d
杨梅素100μmol/L组	0.83±0.01	16.37^b	0.00^b	0.58±0.01	12.01^d	0.00^d

组别	p-Akt蛋白相对灰度值 ( $x ˉ$ ±s)	t值	P值	p-mTOR蛋白相对灰度值 ( $x ˉ$ ±s)	t值	P值
空白组	0.19±0.01			0.27±0.00
模型组30min	1.23±0.01	84.60^a	0.00^a	0.28±0.01	0.60^f	0.58^f
模型组60min	1.52±0.01	66.79^a	0.00^a	0.27±0.01	0.36^f	0.74^f
模型组180min	0.74±0.02	30.49^a	0.00^a	0.57±0.00	94.61^f	0.00^f
空白+杨梅素	0.19±0.00	0.92^b	0.41^b	0.29±0.00	0.91^g	0.41^g
杨梅素30min	0.99±0.01	27.60^c	0.00^c	0.29±0.01	1.31^h	0.26^h
杨梅素60min	0.96±0.01	30.06^d	0.00^d	0.33±0.01	2.31ⁱ	0.08ⁱ
杨梅素180min	0.43±0.01	18.60^e	0.00^e	0.46±0.01	21.60^j	0.00^j

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