Phlorizin allevistes oxidative stress and apoptosis of rat cardiac myocytes H9C2 induced by hypoxia/reoxygenation by down-regulating miR-125a-5p

doi:10.11958/20240846

Abstract

Abstract:

Objective To investigate the effect of phlorizin on the apoptosis and oxidative stress of rat cardiomyocytes H9C2 induced by hypoxia/reoxygenation (H/R) and its possible mechanism. Methods H9C2 cells were cultured in vitro. H/R model was established after pretreatment with different doses (16, 32, 64 μmol/L) of phlorizin or transfection with anti-miR-125a-5p, anti-miR-NC, miR-125a-5p mimics and negative controls. Proliferation was detected by CCK-8, and apoptosis was detected by flow cytometry. The protein expression levels of B lymphoblastoma-2 associated X protein (Bax) and B lymphocytoma-2 (Bcl-2) were detected by Western blot assay. The release of lactate dehydrogenase (LDH) and the activity of superoxide dismutase (SOD) were detected by colorimetric method. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of miR-125a-5p. Results Compared with the H/R group, inhibition rate, apoptosis rate, Bax protein expression, LDH content and miR-125a-5p expression were decreased after low, medium and high doses of phlorizin treatment (P<0.05), and SOD activity, Bcl-2 protein expression were increased (P<0.05). After inhibiting the expression of miR-125a-5p, the inhibition rate, apoptosis rate, Bax protein expression and LDH content of H9C2 cells induced by H/R were decreased (P<0.05), and SOD activity, Bcl-2 protein expression were increased (P<0.05). Overexpression of miR-125a-5p reversed the effect of phloridin on H/R-induced proliferation, apoptosis and oxidative stress of H9C2 cells. Conclusion Phlorizin may reduce H/R-induced apoptosis and oxidative stress in H9C2 cells by decreasing the expression of miR-125a-5p.

Key words: myocardial reperfusion injury, cell hypoxia, myocardium, microRNAs, apoptosis, oxidative stress, Phlorizin, miR-125a-5p

CLC Number:

R541.1

Figures/Tables 11

References 20

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基因名称	引物序列（5'→3'）	产物大小/bp
miR-125 a-5p	上游：ACGGTGCTGGATGTGG CCTTT	167
	下游：GGCCAACCGCGAGAAGATGT TTTTTTTT	167
U6	上游：GCTAGAGGCGCTGCCGAC	93
	下游：CGTAGAGCTCGCTGGGACG	93

基因名称	引物序列（5'→3'）	产物大小/bp
miR-125 a-5p	上游：ACGGTGCTGGATGTGG CCTTT	167
	下游：GGCCAACCGCGAGAAGATGT TTTTTTTT	167
U6	上游：GCTAGAGGCGCTGCCGAC	93
	下游：CGTAGAGCTCGCTGGGACG	93

组别		LDH/（U/L）	Bax	Bcl-2
con组		129.44±15.66	0.13±0.01	0.78±0.06
H/R组		535.55±25.62^a	0.67±0.05^a	0.18±0.02^a
H/R+根皮苷L组		444.65±27.05^b	0.52±0.04^b	0.29±0.03^b
H/R+根皮苷M组		331.67±23.14^bc	0.38±0.03^bc	0.46±0.04^bc
H/R+根皮苷H组		251.73±17.65^bcd	0.24±0.02^bcd	0.56±0.05^bcd
F		459.825^＊＊	379.391^＊＊	274.400^＊＊
组别	抑制率/%		凋亡率/%	SOD/（U/L）
con组	0.00±0.00		6.59±0.28	338.86±22.03
H/R组	54.92±2.25^a		23.64±1.12^a	57.80±4.60^a
H/R+根皮苷L组	44.76±2.34^b		19.84±0.87^b	102.40±10.39^b
H/R+根皮苷M组	33.38±1.72^bc		15.10±0.85^bc	187.27±9.09^bc
H/R+根皮苷H组	24.14±1.67^bcd		11.39±0.78^bcd	256.77±17.38^bcd
F	1 225.229^＊＊		596.470^＊＊	582.878^＊＊

组别		LDH/（U/L）	Bax	Bcl-2
con组		129.44±15.66	0.13±0.01	0.78±0.06
H/R组		535.55±25.62^a	0.67±0.05^a	0.18±0.02^a
H/R+根皮苷L组		444.65±27.05^b	0.52±0.04^b	0.29±0.03^b
H/R+根皮苷M组		331.67±23.14^bc	0.38±0.03^bc	0.46±0.04^bc
H/R+根皮苷H组		251.73±17.65^bcd	0.24±0.02^bcd	0.56±0.05^bcd
F		459.825^＊＊	379.391^＊＊	274.400^＊＊
组别	抑制率/%		凋亡率/%	SOD/（U/L）
con组	0.00±0.00		6.59±0.28	338.86±22.03
H/R组	54.92±2.25^a		23.64±1.12^a	57.80±4.60^a
H/R+根皮苷L组	44.76±2.34^b		19.84±0.87^b	102.40±10.39^b
H/R+根皮苷M组	33.38±1.72^bc		15.10±0.85^bc	187.27±9.09^bc
H/R+根皮苷H组	24.14±1.67^bcd		11.39±0.78^bcd	256.77±17.38^bcd
F	1 225.229^＊＊		596.470^＊＊	582.878^＊＊

组别	抑制率/%		凋亡率/%		SOD/（U/L）
H/R+anti-miR-NC组	54.56±2.60		23.50±1.68		58.20±5.22
H/R+anti-miR-125a-5p组	14.50±1.05		9.61±0.65		270.28±24.92
t	42.860^＊＊		23.133^＊＊		24.989^＊＊
组别		LDH/（U/L）		Bax		Bcl-2
H/R+anti-miR-NC组		541.67±29.72		0.67±0.07		0.17±0.02
H/R+anti-miR-125a-5p组		196.60±12.91		0.18±0.02		0.63±0.05
t		31.948^＊＊		20.192^＊＊		25.626^＊＊