Astragaloside IV suppresses post-ischemic natural killer cell infiltration and activation in the brain: involvement of histone deacetylase inhibition

doi:10.1007/s11684-020-0783-8

Front. Med.

2021, Vol. 15

Issue (1) : 79-90 https://doi.org/10.1007/s11684-020-0783-8

RESEARCH ARTICLE

Astragaloside IV suppresses post-ischemic natural killer cell infiltration and activation in the brain: involvement of histone deacetylase inhibition

Baokai Dou^1,², Shichun Li^1,³, Luyao Wei^1,³, Lixin Wang¹, Shiguo Zhu¹, Zhengtao Wang², Zunji Ke³, Kaixian Chen²(

), Zhifei Wang^1,³(

)

¹. School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
². Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
³. Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

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Abstract

Natural killer (NK) cells, a type of cytotoxic lymphocytes, can infiltrate into ischemic brain and exacerbate neuronal cell death. Astragaloside IV (ASIV) is the major bioactive ingredient of Astragalus membranaceus, a Chinese herbal medicine, and possesses potent immunomodulatory and neuroprotective properties. This study investigated the effects of ASIV on post-ischemic brain infiltration and activation of NK cells. ASIV reduced brain infarction and alleviated functional deficits in MCAO rats, and these beneficial effects persisted for at least 7 days. Abundant NK cells infiltrated into the ischemic hemisphere on day 1 after brain ischemia, and this infiltration was suppressed by ASIV. Strikingly, ASIV reversed NK cell deficiency in the spleen and blood after brain ischemia. ASIV inhibited astrocyte-derived CCL2 upregulation and reduced CCR2⁺ NK cell levels in the ischemic brain. Meanwhile, ASIV attenuated NK cell activating receptor NKG2D levels and reduced interferon-γ production. ASIV restored acetylation of histone H3 and the p65 subunit of nuclear factor-κB in the ischemic brain, suggesting inhibition of histone deacetylase (HDAC). Simultaneously, ASIV prevented p65 nuclear translocation. The effects of ASIV on reducing CCL2 production, restoring acetylated p65 levels and preventing p65 nuclear translocation were mimicked by valproate, an HDAC inhibitor, in astrocytes subjected to oxygen-glucose deprivation. Our findings suggest that ASIV inhibits post-ischemic NK cell brain infiltration and activation and reverses NK cell deficiency in the periphery, which together contribute to the beneficial effects of ASIV against brain ischemia. Furthermore, ASIV’s effects on suppressing NK cell brain infiltration and activation may involve HDAC inhibition.

Keywords astragaloside IV brain ischemia natural killer cells histone deacetylase nuclear factor-κB

Corresponding Author(s): Kaixian Chen,Zhifei Wang

Just Accepted Date: 15 September 2020 Online First Date: 17 December 2020 Issue Date: 11 February 2021

Cite this article:

Baokai Dou,Shichun Li,Luyao Wei, et al. Astragaloside IV suppresses post-ischemic natural killer cell infiltration and activation in the brain: involvement of histone deacetylase inhibition[J]. Front. Med., 2021, 15(1): 79-90.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-020-0783-8
https://academic.hep.com.cn/fmd/EN/Y2021/V15/I1/79

Fig.1 ASIV improved functional deficits and reduced cerebral infarction in MCAO rats. (A) The chemical structure of ASIV. (B) ASIV at 20 mg/kg robustly increased the rotarod retention time of MCAO rats, and this effect persisted for at least 7 days after MCAO. (C) ASIV at 5, 10, and 20 mg/kg markedly reduced neurological deficit score of MCAO rats on day 1, and ASIV at the highest dosage consistently provided protection for at least 7 days. (D) ASIV dose-dependently reduced brain infarct volume in MCAO rats on day 7. ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. sham; *P<0.05, **P<0.01, ***P<0.001 vs. MCAO; n = 8.

Parameter	Day	Sham	MCAO	MCAO+ 5 mg/kg ASIV	MCAO+ 10 mg/kg ASIV	MCAO+ 20 mg/kg ASIV
Max contact area (mm²)	1	118.2±12.0	56.6±9.3^###	77.3±12.4	97.8±10.2*	97.8±6.7**
	3	110.1±8.4	78.4±5.8^#	95.7±10.1	102.1±6.5	105.3±7.6*
	7	123.5±5.3	71.8±10.6^###	92.2±10.8	80.9±9.7	107.5±6.6**
Max contact max intensity	1	188.1±6.1	134.5±12.6^##	155.9±11.4	175.7±9.5	176.9±9.1*
	3	199.9±4.1	165.5±10.9^#	181.7±18.8	190.3±2.7	193.0±5.8*
	7	203.0±2.2	166.6±16.2^##	184.4±7.7	176.1±12.7	197.9±2.3*
Max contact mean intensity	1	85.1±3.2	64.4±3.1^##	72.9±3.0	79.7±4.2	78.1±3.7*
	3	86.2±2.3	74.4±2.4^#	81.0±4.5	82.9±1.6	84.9±2.6*
	7	88.7±2.6	73.9±4.2^##	78.9±3.2	76.3±3.8	84.9±1.9*
Print width (mm)	1	20.2±0.7	12.2±0.9^###	13.2±1.6	15.0±0.6	16.4±0.6**
	3	19.1±0.6	16.1±0.7	17.8±1.5	17.2±0.7	18.4±0.4
	7	21.6±1.0	17.9±0.8	20.1±0.6	19.1±0.3	19.7±0.6
Print area (mm²)	1	133.2±5.1	75.8±11.1^#	90.5±13.8	115.5±9.9	125.0±12.4*
	3	131.6±9.6	91.5±6.1	109.3±10.6	118.2±8.0	119.7±8.2
	7	151.2±15.0	96.7±11.2	111.7±11.6	94.3±11.0	128.1±8.8
Mean intensity	1	87.9±2.6	67.4±2.7^##	74.8±3.5	83±4.4*	81.4±3.8*
	3	90.6±2.3	79.7±2.1^#	85.0±5.3	87.5±1.7	89.6±2.8*
	7	92.9±2.3	77.6±4.6^##	83.1±3.2	80.1±4.1	89.4±1.9*
Swing speed (mm/s)	1	964.5±75.4	568.1±86.1^##	846.6±147.1	845.6±22.0*	893.2±54.5**
	3	1011.2±73.1	809.8±82.2	1036.1±62.7	952.7±60.5	929.0±50.7
	7	1024.5±111.8	897.7±46.1	1097.1±60.1	852.4±60.8	958.9±37.4
Stride length (mm)	1	123.0±5.5	86.0±3.5^###	93.9±5.6	102.0±7.8	110.6±5.4**
	3	128.8±7.1	117.0±7.9	132.1±6.1	132.1±6.4	123.0±4.9
	7	119.8±5.4	128.9±5.1	132.4±5.9	114.9±8.2	133.9±5.8
Duration (s)	1	1.2±0.1	2.5±0.2^###	2.2±0.2	1.8±0.2	1.6±0.1**
	3	1.2±0.2	1.8±0.2	1.1±0.1	1.3±0.1	1.5±0.2
	7	1.4±0.2	1.3±0.1	1.2±0.1	1.4±0.1	1.2±0.1
Average speed (mm/s)	1	343.6±24.4	158.1±16.9^###	178.5±16.5	232.9±21.3	258.4±25.5*
	3	319.1±29.7	253.7±32.1	360.9±18.8	318.8±30.4	292.3±21.7
	7	298.4±28.4	310.5±24.3	352.4±24.4	297.1±16.5	334.3±20.0

Tab.1 ASIV ameliorated gait deficits in MCAO rats

Fig.2 ASIV suppressed brain infiltration of NK cells and reversed NK cell deficiency in the periphery after brain ischemia. (A) As assessed by immunohistochemistry, abundant CD161⁺ cells were accumulated in the ischemic cortex and this accumulation was robustly inhibited by ASIV. (B) Flow cytometry revealed that ASIV markedly suppressed the infiltration of NK cells (CD161⁺CD3⁻) into the ischemic hemisphere. Simultaneously, the NK cell levels were significantly decreased in the spleen and blood after brain ischemia, which were restored by ASIV treatment. ***P<0.001, **P<0.01, *P<0.05, n = 8.

Fig.3 ASIV downregulated CCL2 and CCR2 in the ischemic brain and on brain-infiltrated NK cells, respectively. (A) ASIV significantly reduced MCAO-induced upregulation of CCL2 mRNA levels. (B) Abundant CCL2 was observed in the ischemic cortex and this upregulation was markedly suppressed by ASIV. (C) CCL2 was colocalized with astrocytes in the ischemic bran. (D) ASIV markedly suppressed CCR2⁺ NK cell levels in the ischemic brain. ***P<0.001, **P<0.01, *P<0.05, n = 8.

Fig.4 ASIV decreased IFN-g production in the ischemia cortex and NKG2D expression on brain-infiltrated NK cells. (A, B) Both mRNA and protein levels of IFN-g were markedly increased in the ischemic cortex and were robustly reduced by ASIV. (C) NKG2D⁺ NK cell levels were increased in the ischemic brain and were significantly reduced by ASIV. ***P<0.001, **P<0.01, *P<0.05, n = 8.

Fig.5 ASIV increased acetylation of histone H3 and p65, and inhibited p65 nuclear translocation in the ischemic brain. (A) ASIV restored ac-H3 levels in the ischemic cortex. (B) ASIV significantly increased ac-p65 levels in the ischemic cortex. (C, D) MCAO-induced nuclear translocation of p65 was markedly inhibited by ASIV, as assessed by Western blotting and immunofluorescent staining, respectively. ***P<0.001, **P<0.01, *P<0.05, n = 8.

Fig.6 ASIV and VPA reduced CCL2 expression, restored p65 acetylation, and inhibited p65 nuclear translocation in OGD-treated primary cultures of rat cortical astrocyte. (A–C) OGD-induced upregulation in CCL2 mRNA levels and protein expression in astrocytes was reduced by ASIV and VPA. (D) ASIV and VPA restored the acetylation of p65 in OGD-treated astrocytes. (E) ASIV and VPA inhibited the nuclear translocation of p65 in OGD-treated astrocytes. ***P<0.001, **P<0.01, *P<0.05, n = 3.

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