雷诺嗪预处理对缺氧/复氧损伤H9C2心肌细胞自噬的调控及对心肌细胞的保护作用

doi:10.13481/j.1671-587x.20200623

Abstract

Abstract: Objective

To explore the protective effect of ranolazine preconditioning on the H9C2 cardiomyocytes injured by hypoxia/reoxygenation， and to elucidate the mechanism of ranolazine in protecting myocardium of ischemia-reperfusion injury of the point of regulation autophagy view.

Methods

The H9C2 cells were randomly divided into normal control group， hypoxia/reoxygenation model group， ranolazine preconditioning group， ranolazine+Wortmannin group， and rapamycin group. The cells in normal control group didn’t receive any treatment， and the H9C2 cells in the other groups were pretreated with the corresponding drugs， and then were treated with hypoxia for 4 h and reoxygenation for 3 h. The viabilities of the H9C2 cells in various groups were detected by CCK-8 method； the levels of lactate dehydrogenase （LDH） in the H9C2 cells in various groups were detected by LDH kit； the positive expression rates of microtubule-associated protein light chain 3（LC3） in the H9C2 cells in various groups were analyzed by immunofluorescence method； Western blotting method was used to detect the expression level of LC3， mammalian target of rapamycin（ mTOR） and phosphorylated mTOR（ p-mTOR） in the H9C2 cells in various groups，　and the ratios of LC3Ⅱ/LC3Ⅰ and p- mTOR/ mTOR were calculated.

Results

Compared with hypoxia/reoxygenation model group， the viabilities of H9C2 cells in ranolazine precoditioning group and rapamycin group were increased significantly （P<0.01）， the activities of LDH in the H9C2 cells were decreased significantly （ P<0.05）， and the positive expression rates of LC3 were increased significantly （ P<0.05）.The Western blotting results showed that compared with normal control group， the ratio of LC3 Ⅱ/LC3Ⅰ in the H9C2 cells in hypoxia/reoxygenation model group was increased significantly （P<0.01） and the ratio of p-mTOR/ mTOR was decreased significantly （P<0.01）.Compared with hypoxia/reoxygenation model group， the ratios of LC3 Ⅱ/LC3Ⅰ in the H9C2 cells in ranolazine preconditioning group and rapamycin group were increased significantly （P<0.01）， and the ratios of p-mTOR/mTOR were decreased significantly （P<0.01）.

Conclusion

Ranolazine preconditioning has a protective effect on the H9C2 cells injured by hypoxia/reoxygenation，and its mechanism may be related to its inhibition in mTOR protein phosphorylation and induction of the autophagy of H9C2 cells injured by hypoxia/ reoxygenation.

Key words: ischemia reperfusion, ranolazine, mamalian target of rapamycin, autophagy, microtubule-associated protein light chain 3

CLC Number:

R329.28

Xi ZHAO,Mingyuan LIU,Meng LI,Haiyan LUAN,Yu YANG,Chunyan MA,Baocheng ZHANG,Jincheng ZHAO,Guangyuan YANG. Regulation of ranolazine preconditioning on autophagy of H9C2 cardiomyocytes injured by hypoxia/reoxygenation and its protective effect on cardiomyocytes[J].Journal of Jilin University(Medicine Edition), 2020, 46(6): 1254-1259.

Figures/Tables 4

Tab.1

Tab.2

Fig.2

Tab.3

References 24

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Group	Viability of H9C2 cells	Cell viability of hypoxia/reoxygenation H9C2 cells
Normal control	1.48 ±0.07	0.85 ±0.07
Hypoxia/reoxygenation model	－	0.50 ±0.01^*
Ranolazine(μmol·L^－1)
2	1.47±0.02	0.52 ±0.04
5	1.39 ±0.05	0.58 ±0.07
10	1.39 ±0.06	0.62 ±0.06^△
15	1.42 ±0.04	0.67 ±0.02^△
20	1.45 ±0.05	0.71 ±0.04^△
30	1.43 ±0.09	0.64 ±0.04^△

Group	Cell viability	LDH activity [λ_B/(U·L^－1)]
Normal control	0.77±0.03	123.87±21.09
Hypoxia/reoxygenation model	0.48±0.02^*	320.77±34.75^*
Ranolazine preconditioning	0.69±0.05^△	210.91±16.93^△
Ranolazine+wortmannin	0.52±0.03	295.45±11.84
Rapamycin	0.66±0.03^△	227.79±18.39^△

Group	LC3 Ⅱ/LC3 Ⅰ	p-mTOR/mTOR (AU)
Normal control	0.94±0.02	1.52±0.06
Hypoxia/reoxygenation model	1.34±0.08^*	1.11±0.05^*
Ranolazine preconditioning	1.93±0.21^△	0.64±0.08^△
Ranolazine+wortmannin	1.04±0.22	1.17±0.08
Rapamycin	2.14±0.11^△	0.33±0.05^△

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Regulation of ranolazine preconditioning on autophagy of H9C2 cardiomyocytes injured by hypoxia/reoxygenation and its protective effect on cardiomyocytes

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