Effects of total flavonids of astragalus on hypoxia/reoxygenation-induced cardiomyocyte injury by regulating miR-190a-5p and its molecular mechanism

doi:10.3877/cma.j.issn.2095-1221.2022.06.004

Abstract

Abstract:

Objective

To investigate the effect of total flavonids of astragalus (TFA) on hypoxia/reoxygenation (H/R) -induced cardiomyocyte injury and its possible mechanism.

Methods

Rat cardiomyocytes H9c2 were divided into a Control group (blank culture) , H/R group (hypoxia/reoxygenation treatment) , H/R+TFA-L group (TFA concentration 50 μg/mL treatment, H/R treatment) , H/R+TFA-M group (TFA concentration of 100 μg/mL treatment, H/R treatment) , H/ R+TFA-H group (TFA concentration of 150 μg/mL treatment, H/R treatment) , H/R+miR-NC group (transfected with miR-NC, H/R treatment) , H/R+miR-190a-5p group (transfected with miR-190a-5p and treated with H/R) , H/R+TFA-H+anti-miR-NC group (transfected with anti-miR-NC, TFA concentration of 100 μg/mL treatment, H/R treatment) , H/R+TFA-H+anti-miR-190a-5p group (transfected with anti-miR-190a-5p, treated with TFA concentration of 100 μg/mL, and performed H/ R treatment) . RT-PCR detection of miR-190a-5p expression; the apoptosis rate was detected by flow cytometry. The kit was used to detect the levels of SOD, MDA, ROS and LDH. Western blot was used to detect the expression of the cleaved caspase-3 protein.

Results

Compared with the Control group, the apoptosis rate of H/R group [ (32.76±2.42) %vs (3.23±0.43) %], cleaved caspase-3 (0.83± 0.05 vs 0.26±0.02) , MDA[ (23.36±2.25) vs (4.26±0.48) nmol/mg], LDH [ (52.51±3.82) vs (13.66±1.72) U/L], ROS [ (192.63±10.42) %vs (98.51±6.55) %] level increased (P < 0.05) , BCL2 protein level (0.33±0.06 vs 0.91±0.05) , SOD [ (52.14±4.95) vs (135.32±9.22) U/mg] level, miR-190a-5p expression (0.32 ± 0.05 vs 1.02 ± 0.10) decreased (P < 0.05) ; compared with H/R group, H/R+TFA-L group, H/R+TFA-M group, H/R+TFA-H group cell apoptosis rate [ (27.09± 2.11) %, (19.21±1.59) %, (11.01±1.25) %vs (32.76±2.42) %], cleaved caspase-3, MDA [ (18.17±1.67) , (14.08±1.22) , (9.84±0.73) vs (23.36±2.25) nmol/ mg], LDH [ (43.11± 4.30) , (31.25±2.59) , (19.42±1.89) vs (52.51 ± 3.82) U/L], ROS [ (166.11± 9.88) %, (141.52±8.94) %, (115.32±7.55) %vs (192.63±10.42) %] level decreased (P < 0.05) , BCL2 protein levels, SOD [ (72.33±5.87) , (92.08±6.27) , (116.38±7.66) vs (52.14± 4.95) U/ mg] level and miR-190a-5p expression increased (P < 0.05) ; compared with H/ R+ miR-NC group, the apoptosis rate [ (13.51±1.65) %vs (33.07±2.52) %], cleaved caspase-3, MDA [ (13.15± 1.67) nmol/mg vs (24.08±3.24) nmol/mg], LDH [ (29.48±2.55) vs (50.68± 4.83) U/ L], ROS [ (132.14±9.47) %vs (180.11±12.45) %] levels in H/R+miR-190a-5p group decreased (P < 0.05) , BCL2 protein levels, SOD [ (85.64±6.11) vs (51.27±4.98) U/mg] level increased (P < 0.05) . Transfection of anti-miR-190a-5p could reduce the effect of TFA on H/R-induced apoptosis and oxidative stress in H9c2 cells.

Conclusion

TFA could inhibit apoptosis and oxidative stress by up-regulating the expression of miR-190a-5p, thereby reducing H/R-induced H9c2 cell damage.

Key words: Total flavonids of astragalus, miR-190a-5p, Hypoxia/reoxygenation, Cardiomyocytes H9c2, Rat, Apoptosis

Pengfei Tian, Lijuan Wang, Shengchao Xiao. Effects of total flavonids of astragalus on hypoxia/reoxygenation-induced cardiomyocyte injury by regulating miR-190a-5p and its molecular mechanism[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(06): 346-352.

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Figures/Tables 9

References 23

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基因	序列（5'-3'）	预期扩增产物长度（bp）
miR-190a-5p	上游GCAGGCCTCTGTGTGATATGT	236
	下游GGCAAGACACTGTAGGAATATGT
U6	上游ATTGGAACGATACAGAGAAGATT	218
	下游GGAACGCTTCACGAATTTG

基因	序列（5'-3'）	预期扩增产物长度（bp）
miR-190a-5p	上游GCAGGCCTCTGTGTGATATGT	236
	下游GGCAAGACACTGTAGGAATATGT
U6	上游ATTGGAACGATACAGAGAAGATT	218
	下游GGAACGCTTCACGAATTTG

分组	凋亡率（%）	cleaved caspase-3	BCL2
对照	3.23±0.43	0.26±0.02	0.91±0.05
H/R	32.76±2.42^a	0.83±0.05^a	0.33±0.06^a
H/R+TFA-L	27.09±2.11^ab	0.71±0.06^ab	0.48±0.05^ab
H/R+TFA-M	19.21±1.59^abc	0.57±0.04^abc	0.65±0.05^abc
H/R+TFA-H	11.01±1.25^bcd	0.42±0.04^abcd	0.81±0.06^bcd
F值	145.738	79.283	57.020
P值	< 0.001	< 0.001	< 0.001

分组	凋亡率（%）	cleaved caspase-3	BCL2
对照	3.23±0.43	0.26±0.02	0.91±0.05
H/R	32.76±2.42^a	0.83±0.05^a	0.33±0.06^a
H/R+TFA-L	27.09±2.11^ab	0.71±0.06^ab	0.48±0.05^ab
H/R+TFA-M	19.21±1.59^abc	0.57±0.04^abc	0.65±0.05^abc
H/R+TFA-H	11.01±1.25^bcd	0.42±0.04^abcd	0.81±0.06^bcd
F值	145.738	79.283	57.020
P值	< 0.001	< 0.001	< 0.001

分组	实验次数	SOD （U/mg）	MDA （nmol/mg）	LDH （U/L）	ROS荧光强度（%）
对照	3	135.32±9.22	4.26±0.48	13.66±1.72	98.51±6.55
H/R	3	52.14±4.95^a	23.36±2.25^a	52.51±3.82^a	192.63±10.42^a
H/R+TFA-L	3	72.33±5.87^ab	18.17±1.67^ab	43.11±4.30^ab	166.11±9.88^ab
H/R+TFA-M	3	92.08±6.27^abc	14.08±1.22^abc	31.25±2.59^abc	141.52±8.94^abc
H/R+TFA-H	3	116.38±7.66^bcd	9.84±0.73^abcd	19.42±1.89^bcd	115.32±7.55^abcd
F值		68.708	80.604	84.136	55.808
P值		< 0.001	< 0.001	< 0.001	< 0.001

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