Allium mongolicum Regel Flavonoids Alleviate Hypoxia/ Reoxygenation-Induced Cardiomyocyte Injury by Downregulating lncRNA TALNEC2

Huaming Zhang; Xi Feng

doi:10.36468/pharmaceutical-sciences.1273

All submissions of the EM system will be redirected to Online Manuscript Submission System. Authors are requested to submit articles directly to Online Manuscript Submission System of respective journal.

Research Paper

Allium mongolicum Regel Flavonoids Alleviate Hypoxia/ Reoxygenation-Induced Cardiomyocyte Injury by Downregulating lncRNA TALNEC2

Huaming Zhang and Xi Feng^*

Department of Cardiology, Clinical Cardiovascular Center, Liyuan Hospital, Tongj Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430071, China

*Corresponding Author:: Xi Feng
Department of Cardiology, Clinical Cardiovascular Center, Liyuan Hospital, Tongj Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430071, China
E-mail: hmz20913@163.com

Date of Received	04 February 2023
Date of Revision	21 September 2023
Date of Accepted	10 February 2024
Indian J Pharm Sci 2024;86(1):266-271

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

DOI: 10.36468/pharmaceutical-sciences.1273

Abstract

To explore the effect and possible mechanism of Allium mongolicum Regel flavonoids on cardiomyocyte injury induced by hypoxia/reoxygenation. H9C2 cardiomyocytes were induced by hypoxia/reoxygenation and treated with different doses of Allium mongolicum Regel flavonoids. H9C2 cells were transfected with si-negative control/ si-TALNEC2 and treated with hypoxia/reoxygenation. Besides, cells transfected with plasmid cloning deoxyribonucleic acid/plasmid cloning deoxyribonucleic acid-TALNEC2 were treated with Allium mongolicum Regel flavonoids and induced with hypoxia/reoxygenation. Malondialdehyde, glutathione peroxidase and superoxide dismutase levels were tested to evaluate oxidative stress. Apoptosis rate was analyzed by flow cytometry. TALNEC2 expression was examined using quantitative reverse transcription-polymerase chain reaction, and cleaved caspase-3 and cleaved caspase-9 protein levels were tested by Western blot. Allium mongolicum Regel flavonoids could reduce malondialdehyde level, apoptosis rate, cleaved caspase-3 level, cleaved caspase-9 level, and TALNEC2 expression, while enhanced glutathione peroxidase and superoxide dismutase levels in hypoxia/reoxygenation-induced H9C2 cells in a dose-dependent manner. After transfection of si-TALNEC2, malondialdehyde level, apoptosis rate, cleaved caspase-3 level, and cleaved caspase-9 level were reduced, while superoxide dismutase and glutathione peroxidase levels were enhanced. Transfection of plasmid cloning deoxyribonucleic acid-TALNEC2 could abolish the effect of Allium mongolicum Regel flavonoids on cardiomyocyte injury. Allium mongolicum Regel flavonoids could inhibit hypoxia/reoxygenation-induced cardiomyocyte apoptosis and oxidative stress via reducing TALNEC2 expression.

Keywords

Allium mongolicum Regel flavonoids, hypoxia/reoxygenation, TALNEC2, cardiovascular disease, malondialdehyde

The mortality of cardiovascular disease is increasing year by year in China[1,2]. Although percutaneous coronary intervention and other treatments have achieved good results, reperfusion therapy can aggravate myocardial tissue damage and cause arrhythmia and other side effects[3,4]. Oxidative stress and apoptosis can cause myocardial Ischemia-Reperfusion (I/R) injury[5,6]. Active ingredients of Traditional Chinese Medicine (TCM) have anti-apoptosis and anti-oxidative stress effects, and can be used to alleviate myocardial I/R injury[7,8]. Therefore, it is of great significance to find effective TCM active ingredients and reveal their potential molecular mechanisms for improving myocardial I/R injury.

Allium mongolicum Regel, belongs to Allium genus of Liliaceae, contains many active ingredients and have certain medicinal value[9]. Studies have shown that Allium mongolicum Regel Flavonoids (AMRF) can promote the contraction of intestinal smooth muscle and improve constipation in mice[10]. Importantly, AMRF has been confirmed to have anti-oxidant, anti-apoptosis and anti-inflammatory properties[11-13]. However, whether AMRF can improve myocardial I/R injury by suppressing cardiomyocyte apoptosis and oxidative stress is still unknown.

Long noncoding RNA (lncRNA) has been confirmed to be involved in human diseases development[14,15]. Previous study suggested that lncRNA TALNEC2 knockdown alleviated cerebral I/R injury via inhibiting neuronal apoptosis and inflammation[16,17]. Moreover, TALNEC2 was overexpressed in myocardial ischemic patients, and its overexpression could promote Hypoxiainduced Cardiomyocytes (H9C2) injury[18]. Here, we found that AMRF exerted an inhibitory effect on TALNEC2 expression. However, whether AMRF can improve myocardial I/R injury through regulating TALNEC2 expression is unclear.

Based on the above, our study investigated whether AMRF affected myocardial I/R injury via regulating TALNEC2 using Hypoxia/Reoxygenation (H/R)- induced H9C2 cells.

Materials and Methods

Preparation of AMRF:

Allium mongolicum Regel (Sihehui Trading, Inner Mongolia, China) was extracted by 75 % ethanol for 2 h (70°), and then the supernatant was obtained by centrifugation. The supernatant was concentrated under the reduced pressure by a rotary evaporator. Sodium hydroxide reaction method was used to determine the composition of flavonoids in the extract (obtained 12.96 mg/g AMRF). AMRF was diluted by Dimethylsulfoxide (DMSO) to prepare different concentrations.

Cell culture and grouping:

H9C2 cells (Procell, Wuhan, China) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10 % Fetal Bovine Serum (FBS). To construct H/R cell model, H9C2 cells were cultured under hypoxia condition (5 % Carbon dioxide (CO₂), 95 % Nitrogen (N₂) and 0.1 % Oxygen (O₂)) for 6 h and then performed reoxygenation (5 % CO₂ and 95 % air) for 12 h[19]. Normal cultured cells were used as control group. H9C2 cells were treated with different concentrations (25, 50, and 100 μg/ ml) of AMRF for 24 h and then induced with H/R, which were recorded as H/R+low-AMRF group, H/R+middle-AMRF group and H/R+high-AMRF group, respectively. H9C2 cells were transfected with si-NC/si-TALNEC2 using Lipofectamine 3000 (Invitrogen, Carlsbad, California, United states of America (USA)) and then induced with H/R, which were recorded as H/R+si-NC group and H/R+si-TALNEC2 group. Also, H9C2 cells transfected with plasmid cloning deoxyribonucleic acid (pcDNA)/pcDNA-TALNEC2 were treated with 100 μg/ml AMRF and induced with H/R, which were recorded as H/R+high-AMRF+pcDNA group and H/R+high-AMRF+pcDNA-TALNEC2 group.

Assessing of oxidative str ess:

H9C2 cells were collected and lysed by repeated freeze-thaw method. Malondialdehyde (MDA), Glutathione Peroxidase (GSh-Px) and Superoxide Dismutase (SOD) levels were detected by corresponding kits according to kit instructions.

Flow cytometry:

H9C2 cells were digested to collect cell suspensions. After suspended with binding buffer, cells were stained with Annexin V-Fluorescein Isothiocyanate (FITC) and Propidium Iodide (PI) (Beyotime, Shanghai, China), and cell apoptosis rate was detected by FACS Calibur flow cytometry.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR):

Total Ribonuclic Acid (RNA) was extracted and complementary DNA (cDNA) was synthesized. qRT-PCR was amplified using SYBR Green (Invitrogen), cDNA and specific primers of TALNEC2. Relative expression was calculated by 2^−ΔΔCt method.

Western blot:

Radioimmunoprecipitation Assay (RIPA) buffer was used to extract total protein. Protein was taken for Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) reaction and Polyvinylidene Difluoride (PVDF) membrane transferring. Membrane was incubated with anti-cleaved caspase-3 (ab90437; 1:1000), anticleaved caspase-9 (1:1000), anti-Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) (1:2500, ab9485), and secondary antibody (1:50000, ab205718). Protein bands were visualized by Enhanced Chemiluminescence (ECL) reagent (Beyotime) and quantitatively analyzed by Quantity One^® software.

Statistical analysis:

Data were expressed as x̄ ±s and analyzed by Statistical Package for the Social Sciences (SPSS) 21.0 software. Student’s t-test and Analysis of Variance (ANOVA) were used for comparisons. p<0.05 was considered significant difference.

Results and Discussion

MDA level was enhanced, while GSH-Px and SOD levels were suppressed in the H/R group (Table 1). Furthermore, MDA level was reduced, while GSH-Px and SOD levels were increased in the H/R+low-AMRF, H/R+middle-AMRF and H/ R+high-AMRF groups (Table 1).

Group	MDA (nmol/l)	SOD (U/ml)	GSH-Px (U/ml)
Control	5.62±0.49	68.44±5.92	82.78±6.86
H/R	45.46±4.29*	21.22±2.12*	33.24±3.02*
H/R+low-AMRF	31.61±3.13^#	34.25±3.37^#	45.03±4.08^#
H/R+middle-AMRF	19.91±1.77^#&	46.16±4.41^#&	60.02±4.09^#&
H/R+high-AMRF	9.87±0.86^#&$	57.65±5.52^#&$	74.42±6.65^#&$
F	369.497	155.151	139.85
p	0.000	0.000	0.000

Table 1: Effects of AMRF on H/R-Induced Cell Oxidative Stress

Apoptosis rate, cleaved caspase-3 and cleaved caspase-9 levels were enhanced in the H/R group (fig. 1A), while were reduced in the H/R+low- AMRF, H/R+middle-AMRF and H/R+high-AMRF groups (Table 2 and fig. 1B).

Fig. 1: Effects of AMRF on H/R-induced apoptosis, (A): Western blot and (B): Flow cytometry

Group	Apoptosis rate (%)	Cleaved caspase-3	Cleaved caspase-9
Control	5.32±0.45	0.25±0.02	0.12±0.02
H/R	32.74±2.94*	0.79±0.06*	0.58±0.04*
H/R+low-AMRF	23.34±2.34^#	0.64±0.05^#	0.42±0.03^#
H/R+middle-AMRF	16.15±1.42^#&	0.51±0.04^#&	0.31±0.03^#&
H/R+high-AMRF	8.61±0.74^#&$	0.34±0.03^#&$	0.18±0.02^#&$
F	329.932	239.650	367.714
p	0.000	0.000	0.000

Table 2: Effects of AMRF on H/R-Induced Apoptosis

TALNEC2 level was enhanced in the H/R group, while was reduced in the H/R+low-AMRF, H/ R+middle-AMRF and H/R+high-AMRF groups (Table 3). MDA level was decreased, while GSH-Px and SOD levels were increased in H/ R+si-TALNEC2 group (Table 4). Apoptosis rate, cleaved caspase-3 and cleaved caspase-9 levels were reduced in H/R+si-TALNEC2 group (Table 5 and fig. 2). As shown in fig. 3 and Table 6, MDA level, apoptosis rate, cleaved caspase-3 and cleaved caspase-9 levels were enhanced, while GSH-Px and SOD levels were decreased in the H/ R+high-AMRF+pcDNA-TALNEC2 group.

Fig. 2: Effects of TALNEC2 knockdown on H/R-induced apoptosis, (A): Western blot and (B): Flow cytometry

Fig. 3: Effects of TALNEC2 overexpression on cell apoptosis, (A): Western blot and (B): Flow cytometry

Group	TALNEC2
Control	1.00±0.00
H/R	3.54±0.27*
H/R+low-AMRF	2.66±0.23^#
H/R+middle-AMRF	1.98±0.12^#&
H/R+high-AMRF	1.36±0.12^#&$
F	302.635
p	0.000

Table 3: Effects of AMRF on TALNEC2 Expression

Group	TALNEC2	MDA (nmol/l)	SOD (U/ml)	GSH-Px (U/ml)
H/R+si-NC	1.00±0.00	48.79±4.41	20.58±2.01	31.54±3.14
H/R+si-TALNEC2	0.32±0.03*	15.54±1.22*	50.31±4.07*	67.07±5.08*
t	68.000	21.800	19.649	17.848
p	0.000	0.000	0.000	0.000

Table 4: Effects of TALNEC2 Knockdown on H/R-Induced Oxidative Stress

Group	Apoptosis rate (%)	Cleaved caspase-3	Cleaved caspase-9
H/R+si-NC	34.23±3.02	0.77±0.04	0.57±0.05
H/R+si-TALNEC2	12.69±1.26*	0.40±0.04*	0.24±0.02*
t	19.748	19.622	18.384
p	0.000	0.000	0.000

Table 5: Effects of TALNEC2 Knockdown on H/R-Induced Apoptosis

Group	TALNEC2	MDA (nmol/l)	SOD (U/ml)	GSH-Px (U/ml)	Apoptosis rate (%)	Cleaved caspase-3	Cleaved caspase-9
H/R+high-AMRF+pcDNA	1.00±0.00	9.51±0.83	59.09±4.71	76.29±6.92	8.26±0.62	0.31±0.03	0.17±0.02
H/R+high-AMRF+pcDNA-TALNEC2	3.26±0.29*	33.24±3.02*	32.75±2.91*	42.99±4.18*	22.04±1.78*	0.68±0.04*	0.47±0.03*
t	23.379	22.730	14.273	13.228	21.932	22.200	24.962
p	0.000	0.000	0.000	0.000	0.000	0.000	0.000

Table 6: Effects of TALNEC2 Overexpression on Cell Injury

Cardiomyocyte ischemia causes oxidative stress, and reperfusion causes cardiomyocyte apoptosis[20,21]. Studies have shown that TCM can inhibit cardiomyocyte apoptosis and oxidative stress by regulating multiple targets[22,23]. lncRNA has been confirmed to be abnormally expressed in myocardial I/R injury[24,25]. However, whether lncRNA can be served as a potential target for TCM to alleviate myocardial I/R injury needs to be further explored.

The polysaccharides and flavonoids of Allium mongolicum Regel may slow down the progression of many diseases[11-13]. Similar to the reports of previous studies[26,27], we found that H/R induction elevated MDA level and decreased GSH-Px and SOD levels in cardiomyocytes, suggesting that H/R induction promoted oxidative stress in cardiomyocytes. Further studies revealed that AMRF reduced MDA level, while enhanced GSHPx and SOD levels in H/R-induced cardiomyocytes, indicating that AMRF could inhibit cardiomyocyte oxidative stress. Besides, H/R induced cardiomyocyte apoptosis, which were consistent with the previously studies[28,29]. Furthermore, H/R-induced apoptosis could be inhibited with the increasing of AMRF concentrations, revealing that AMRF repressed H/R-induced apoptosis in cardiomyocytes.

TALNEC2 was upregulated in cerebral I/R injury mouse models, which promoted neuronal apoptosis to facilitate cell injury[16,17]. Besides, inhibition of TALNEC2 attenuated hypoxia-induced injury in mouse embryonic osteoblasts[30]. Our study revealed that TALNEC2 expression was elevated in H/R-induced cardiomyocytes, and AMRF was able to reduce TALNEC2 expression in a concentration-dependent manner. Furthermore, TALNEC2 knockdown inhibited cardiomyocyte injury, whereas its upregulation attenuated the inhibitory effect of AMRF on cardiomyocyte injury. Here, AMRF mitigated myocardial I/R injury by decreasing TALNEC2 level.

In summary, AMRF inhibited H/R-induced apoptosis and oxidative stress in cardiomyocytes depending on reducing TALNEC2 expression. Our findings confirmed that TALNEC2 might serve as a potential target for AMRF in treating myocardial I/R injury.

Conflict of interests:

The authors declared no conflict of interests.