根皮苷通过下调miR-125a-5p减轻缺氧/复氧诱导的H9C2细胞氧化应激和凋亡

doi:10.11958/20240846

摘要/Abstract

摘要：

目的探讨根皮苷对缺氧/复氧（H/R）诱导的大鼠心肌细胞H9C2凋亡和氧化应激的影响及可能机制。方法体外培养H9C2细胞，用16、32、64 μmol/L根皮苷预处理或转染anti-miR-125a-5p、anti-miR-NC、miR-125a-5p模拟物、模拟物阴性对照后建立H/R模型。CCK-8法测定细胞增殖，流式细胞术评估细胞凋亡，Western blot测定B淋巴细胞瘤-2（Bcl-2）蛋白和Bcl-2相关X（Bax）蛋白表达，比色法检测乳酸脱氢酶（LDH）释放量和超氧化物歧化酶（SOD）活性，实时荧光定量PCR（qRT-PCR）检测细胞中微小RNA-125a-5p（miR-125a-5p）表达。结果与H/R组比较，低、中及高剂量根皮苷处理后细胞抑制率、凋亡率、Bax蛋白表达、LDH含量、miR-125a-5p表达依次降低，SOD活性、Bcl-2蛋白表达依次增加（P<0.05）。抑制miR-125a-5p表达后，H/R处理细胞的抑制率、凋亡率、Bax蛋白表达及LDH含量降低，SOD活性、Bcl-2蛋白表达增加（P<0.05）。miR-125a-5p过表达逆转了根皮苷对H/R刺激H9C2细胞凋亡、增殖及氧化应激的作用。结论根皮苷可能通过降低miR-125a-5p表达来减轻H/R诱导的H9C2细胞的氧化应激和凋亡。

关键词: 心肌再灌注损伤, 细胞低氧, 心肌, 微RNAS, 细胞凋亡, 氧化性应激, 根皮苷, miR-125a-5p

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

中图分类号:

R541.1

图/表 11

表1 引物序列

Tab.1 Primers sequence information

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

图1 根皮苷对H/R诱导的H9C2细胞凋亡的影响

Fig.1 Effect of phloridin on H/ R-induced apoptosis of H9C2 cells

图2 根皮苷对H/R诱导的H9C2细胞中Bax和Bcl-2蛋白表达的影响 A：con组；B：H/R组；C：H/R+根皮苷L组；D：H/R+根皮苷M组；E：H/R+根皮苷H组。

Fig.2 Effect of phloridin on expression levels of Bax and Bcl-2 proteins in H/R-induced H9C2 cells

表2 各组氧化应激、凋亡指标及相关蛋白表达水平比较（n=9，$\bar{x}\pm s$）

Tab.2 Comparison of expression levels of oxidative stress, apoptosis and related proteins between the five groups

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^＊＊

图3 根皮苷对H/R诱导的H9C2细胞中miR-125a-5p表达的影响 A：con组；B：H/R组；C：H/R+根皮苷L组；D：H/R+根皮苷M组；E：H/R+根皮苷H组。n=9，F=1 703.769，P<0.05；a与con组比较，b与H/R组比较，c与H/R+根皮苷L组比较，d与H/R+根皮苷M组比较，P<0.05。

Fig.3 Effect of phloridin on the expression of miR-125a-5p in H/ R-induced H9C2 cells

图4 下调miR-125a-5p对H/R诱导的H9C2细胞凋亡的影响

Fig.4 Effect of down-regulation of miR-125a-5p on H/R-induced apoptosis of H9C2 cells

图5 下调miR-125a-5p对H/R诱导的H9C2细胞中Bax和Bcl-2蛋白表达的影响 A：H/R+anti-miR-NC组；B：H/R+anti-miR-125a-5p组。

Fig.5 Effect of down-regulation of miR-125a-5p on expression levels of Bax and Bcl-2 proteins in H/ R-induced H9C2 cells

表3 各组细胞凋亡和氧化应激指标比较（n=9，$\bar{x}\pm s$）

Tab.3 Comparison of apoptosis and oxidative stress indexes in all groups

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^＊＊

图6 上调miR-125a-5p降低根皮苷对H/R诱导的H9C2细胞凋亡的影响

Fig.6 Up-regulation of miR-125a-5p decreased the effect of phloridin on H/R-induced apoptosis of H9C2 cells

图7 上调miR-125a-5p降低根皮苷对H/R诱导的H9C2细胞中Bax和Bcl-2蛋白表达的影响 A：H/R+根皮苷+miR-NC组；B：H/R+根皮苷+miR-125a-5p组。

Fig.7 Up-regulation of miR-125a-5p decreased the effect of phloridin on expression levels of Bax and Bcl-2 proteins in H/R-induced H9C2 cells

表4 2组细胞凋亡和氧化应激指标比较（n=9，$\bar{x}\pm s$）

Tab.4 Comparison of apoptosis and oxidative stress indexes between the two groups

组别	抑制率/%	凋亡率/%	SOD/（U/L）
H/R+根皮苷+miR-NC组	23.97±2.11	11.50±0.81	257.96±22.28
H/R+根皮苷+miR-125a-5p组	48.89±3.24	21.99±0.85	78.14±10.61
t	19.335^＊＊	26.803^＊＊	21.861^＊＊
组别	LDH/（U/L）	Bax	Bcl-2
H/R+根皮苷+miR-NC组	246.94±23.15	0.24±0.03	0.57±0.04
H/R+根皮苷+miR-125a-5p组	497.55±23.24	0.54±0.05	0.24±0.03
t	22.920^＊＊	15.435^＊＊	19.800^＊＊

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