Protective Effects of NaHS/miR-133a-3p on Lipopolysaccharide-Induced Cardiomyocytes Injury

Objective The aim of this study was to investigate the effects of sodium hydrosulfide (NaHS) on Lipopolysaccharide (LPS)-induced cardiomyocyte injury in H9c2 cells. Methods H9c2 cardiomyocytes cultivated with medium containing 10 μg/mL LPS were used to recapitulate the phenotypes of those in sepsis. Two sequential experiments were performed. The first contained a control group, a LPS group, and a LPS + NaHS group, with the aim to assure the protective effects of NaHS on LPS-treated cardiomyocytes. The second experiment added a fourth group, the LPS + NaHS + miR-133a-3p inhibition group, with the aim to preliminarily explore whether miR-133-3p exerts a protective function downstream of NaHS. The adenosine triphosphate (ATP) kit was used to detect ATP content; real-time quantitative polynucleotide chain reaction (qPCR) was used to measure the levels of mammalian targets of rapamycin (mTOR), AMP-dependent protein kinase (AMPK), and miR-133a-3p, and Western blot (WB) was used to detect protein levels of mTOR, AMPK, myosin-like Bcl2 interacting protein (Beclin-1), microtubule-associated protein 1 light chain 3 (LC3I/II), and P62 (sequestosome-1, sqstm-1/P62). Results Compared with the control group, the expressions of miR-133a-3p (P < 0.001), P62 (P < 0.001), and the content of ATP (P < 0.001) decreased, while the expressions of Beclin-1 (P = 0.023) and LC3I/II (P = 0.048) increased in the LPS group. Compared with the LPS group, the expressions of miR-133a-3p (P < 0.001), P62 (P < 0.001), and the content of ATP (P < 0.001) in the NaHS + LPS group increased, while the expressions of Beclin-1 (P = 0.023) and LC3I/II (P = 0.022) decreased. Compared with the NaHS + LPS group, the expression levels of miR-133a-3p (P < 0.001), P62 (P = 0.001), and the content of ATP (P < 0.001) in the LPS + NaHS + miR-133a-3p inhibition group were downregulated, and the expression levels of Beclin-1 (P = 0.012) and LC3I/II (P = 0.010) were upregulated. The difference was statistically significant. There was no significant difference in the expression of AMPK and mTOR between groups. Conclusion Our research demonstrated that NaHS relieved LPS-induced myocardial injury in H9c2 by promoting the expression of miR-133a-3p, inhibiting autophagy in cardiomyocytes, and restoring cellular ATP levels.

However, how sepsis infuences myocardial and underlying mechanisms remains largely unknown.Hydrogen sulfde is an important endogenous biological signaling molecule in mammals and plays an important role in the physiological and pathophysiological processes of the cardiovascular system [4].Sodium hydrosulfde (NaHS) is an important exogenous donor of hydrogen sulfde [5].NaHS is involved in cellular autophagy through multiple pathways, which inhibits autophagy by activation of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mTOR pathway and improves brain function after traumatic brain injury [6].Also, NaHS can protect against septic cardiomyopathy by inhibiting autophagy through the AMPK/ mTOR pathway [7].Terefore, we speculated that NaHS may be involved in the regulation of mTOR signaling.
MicroRNAs (miRNAs) are a class of small endogenous noncoding single-stranded RNAs with a length of about 22 nt.miRNAs play an important regulatory role in the development of many cardiovascular diseases [8,9].Our previous experimental results revealed that miR-133a-3p was diferentially expressed in LPS-treated cardiomyocytes.miR-133a-3p can promote the progression of thyroid cancer by regulating the PI3K/AKT/mTOR pathway [10].Guanidinoacetic acid regulated myogenic diferentiation and muscle growth through the miR-133a-3p and miR-1a-3p comediated Akt/mTOR/S6K signaling pathway [11].Recently, some studies indicated autophagy participated in the molecular mechanism of body damage via measuring miRNAs, ATP, mTOR, AMPK, Beclin-1, LC3I/II, and P62.Cadmium decreased mTOR, increased miR-25-3p, LC3II, and Beclin-1, and caused liver damage and autophagy [12].Energetic impairment as well as miR-9-5p and mTOR decreases occurred in cadmium exposure-caused myocardial injury and autophagy [13].Energy metabolism disorder, mTOR, Beclin-1, LC3b, and P62 took part in the molecular mechanism of chlorpyrifos-induced liver damage and autophagy [14].However, it is an unclear relationship between NaHS and miR-133a-3p in the development of myocardial injury in LPSinduced models.Terefore, this study used LPS-treated H9c2 cells to mimic sepsis-induced myocardial damage [3].We aimed to elucidate the protective efect of NaHS on myocardial injury through miR-133a-3p-regulated autophagy and to provide new insights for basic research and clinical treatment of sepsis-induced myocardial injury.

Grouping and Model
Building.H9c2 cells were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China, CL-0089).H9c2 cells were cultured to logarithmic growth phase and inoculated in 6-well plates with a concentration of 1 × 10 5 /mL.Te cells were cultured overnight, and the old medium was removed when the cells reached a proportion of 70-80% in the wells.Te medium was replaced according to diferent groups (each group contained three samples): (1) control group: cultured in a fresh medium for 24 hours; (2) LPS group: cultured in a medium containing 10 μg/mL of LPS for 24 hours; (3) LPS + NaHS group: cultured in a medium containing 10 μg/ mL of LPS + 200 μM NaHS for 24 hours; and (4) LPS + NaHS + miR-133a-3p inhibition group: in this group, siRNA interference was conducted to inhibit miR-133a-3p expression, and the cells were incubated for 36 hours in a fresh complete medium without double antibodies.Following siRNA transfection, the cells were then treated with 10 μg/mL of LPS + 200 μM NaHS for additional 24 hours.Te concentrations of NaHS and LPS used were selected based on the data from literature and previous experiments [15][16][17].

Cell Transfection. Transfection was performed with
riboFECT ™ CP Transfection Reagent (RiboBio, China, R10035.7).Transfection Reagent was used according to the instructions.A density of 5 × 10 4 to 1 × 10 5 cells was inoculated in 12-well plates containing the appropriate amount of complete medium.Transfection Reagent T was added at a concentration of 100 nM to cells when the cell proportion reached 30-50% of the wells.Te cells were incubated with fresh complete medium without double antibodies at the time of transfection.Te plates were incubated at 37 °C in an incubator with CO 2 for 36 h.Afterwards, the transfected cells were treated with dosing for 24 h.

ATP Assay.
ATP assay kit (Beyotime, China, S0026) was used to perform ATP assay on the cells according to the kit instructions.

RNA Extraction and Real-Time Quantitative PCR.
RNA extraction and real-time quantitative PCR (RT-qPCR) procedures were performed according to the instructions of the kits.Total RNA was isolated from cells using RNAiso (Takara, Japan, D312).mRNA expression was assessed by RT-qPCR using a frst-strand cDNA synthesis kit (Takara, Japan, RR036A) and a qPCR kit (Takara, Japan, RR390A).Te expression levels of miR-133a-3p, mTOR, and AMPK were detected.For miR-133a-3p, U6 was regarded as the internal reference, and GAPDH was regarded as the internal reference for the other genes.Te primer sequences for these genes are listed in Table 1.qPCR conditions were set as follows: 95 °C for 2 min, 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 1 min, with a total of 40 cycles.Triplicate analysis was conducted for all samples.Te relative expression levels of genes were calculated by using the 2 −△△Ct method.

Western Blot (WB).
Te total protein was extracted, and the protein concentration was determined by using a protein concentration detection kit (Takara, Japan, T9300A).Protein samples were mixed with a bufer at a ratio of 4 : 1, and protein concentrations were kept constant at 4 μg/μL.Protein samples were separated using a 4-20% SDS-PAGE gradient prep gel (Kingsley, China), and the separated proteins were transferred onto PVDF membranes.Te primary antibodies of mTOR, AMPK, LC3I/II, P62, and Beclin-1 were diluted with 3% bovine serum albumin at a ratio of 1 : 1000 to 1 : 2000.Tey were purchased from CST (USA).Te membranes were added with primary antibodies and incubated at 4 °C overnight.Te next day, the secondary antibody (diluted to 1 : 5000 with the closure solution, Santa Cruz, USA) was added to the samples for incubating for 1 h.Te enhanced chemiluminescence reagent exposure solution Journal of Toxicology was mixed at a ratio of 1 : 1 of liquid A: liquid B and evenly covered on the membrane for 1 min.Finally, the membrane was examined by using the exposure meter.

Statistical Analysis.
Te data were analyzed using SPSS (version 20.0).All data were presented as mean ± standard deviation (x ± s).Te means of multiple groups were compared by one-way ANOVA, and a LSD-t test was performed between groups.A p value <0.05 was considered as statistically signifcant.

Te Efect of NaHS on ATP and mir-133a-3p Levels in LPS-
Treated Rat Cardiomyocytes.ATP and miR-133a-3p levels were signifcantly lower in the LPS group than those in the control group (P < 0.001 and P � 0.001, respectively).However, ATP and miR-133a-3p levels were signifcantly increased in the LPS + NaHS group compared with those in the LPS group (P < 0.001 and P < 0.001, respectively) (Figure 1).

Te Efect of NaHS on Autophagy-Related Factors in LPS-
Treated Rat Myocardium.Te transcript levels and protein levels of AMPK and mTOR were altered after LPS and NaHS treatment, but the diferences were not statistically signifcant between the groups (P > 0.05).For protein expression levels of Beclin-1, LC3I/II, and P62, we observed increased expression levels of Beclin-1 protein and LC3I/II protein, but decreased expression levels of P62 protein after LPS treatment, compared with the control group (P � 0.023, 0.048, and P < 0.001, respectively).However, in the LPS + NaHS group compared with the LPS group, Beclin-1 protein and LC3I/II protein expressions were signifcantly decreased and P62 protein expression was signifcantly increased (P � 0.023, 0.022, and P < 0.001, respectively) (Figure 2).

NaHS-Regulated ATP Expression via miR-133a-3p.
Compared with the control group, the levels of ATP and miR-133a-3p were decreased in the LPS group (P < 0.001 and P < 0.001, respectively).Compared with the LPS group, ATP and miR-133a-3p expressions were signifcantly increased in the LPS + NaHS group (P < 0.001 and P < 0.001, respectively).

NaHS-Regulated
Autophagy via miR-133a-3p.We found no signifcant diferences in the transcriptional and protein levels of mTOR and AMPK among the control, LPS, LPS + NaHS, and LPS + NaHS + mir-133a-3p groups (P > 0.05 and P > 0.05, respectively), which suggested that AMPK and mTOR may be not involved in autophagy in myocardial damage induced by LPS.Compared with the control group, P62 protein expression was decreased, but Beclin-1 and LC3I/II protein expressions were increased in the LPS group (P < 0.001, P < 0.001, and P � 0.003, respectively).While P62 protein expression was increased, Beclin-1 and LC3I/II proteins were decreased after NaHS treatment compared with the LPS group (P < 0.001, P < 0.001, and P � 0.005).However, the expression of P62, Beclin-1, and LC3I/II protein was reversed (P62 expression was decreased and Beclin-1 and LC3I/II expressions were increased) by miR-133a-3p compared to that in the LPS + NaHS group (P � 0.001, P � 0.012, and P � 0.010, respectively) (Figure 4).

Discussion
ATP is the pivotal energy-carrying molecule within every cell, and the inability to sustain sufcient ATP levels can prove to be a critical factor in numerous diseases [18].In intact HepG2 cells, LPS decreased mitochondrial membrane potential and cellular ATP content [19].Similarly, in mouse bone marrow-derived macrophages activated with LPS, total ATP levels decreased signifcantly [20].In this study, we found that the ATP level was signifcantly lower in the LPS group than that in the control group.H 2 S can provide electrons for mitochondria through the electron transport chain: quinone oxidoreductase (SQR) and mitochondrial 4 Journal of Toxicology complex II, which stimulates oxidative phosphorylation and increases mitochondrial ATP production [21].NaHS acts as a H 2 S donor and can provide H 2 S to organisms for producing the corresponding biological functions.Te present study confrmed that the ATP content of LPS and NaHScotreated rat cardiomyocytes was higher than that of rat cardiomyocytes treated with LPS alone.NaHS can regulate apoptosis and autophagy in various ways.Previous studies have shown that NaHS can inhibit LPS-induced injury in A549 lung cancer cells by increasing cell viability, transmembrane electrical impedance, and expression of occluded small-band proteins and decreasing CRP levels [22].NaHS can restore mitochondrial function and inhibit autophagy through the PI3K/Akt/mTOR signaling pathway to ameliorate traumatic brain injury [23].In addition, NaHS can also activate autophagy to protect colon cells in ulcerative colitis [23] and protect against LPSinduced AKI by promoting autophagy [24].However, what mechanisms of NaHS play an important role in LPSinduced myocardial damage remains unclear.Our study found that P62 protein expression was decreased and the expressions of Beclin-1 and LC3I/II proteins were elevated in rat cardiomyocytes after LPS treatment, whereas increased P62 protein expression and decreased Beclin-1 and LC3I/II protein expression were observed in LPS and NaHScotreated cardiomyocytes with NaHS treatment.Te simultaneous increase in ATP levels was detected.Terefore, we hypothesized that NaHS may improve myocardial injury by reducing ATP consumption.Tese results provide direction for future experiments and further research into the correlation between ATP and autophagy.
Te core lipid kinase complex of autophagy contains BECN1, phosphatidylinositol-3-kinase catalytic subunit type 3 (PIK3C3), and phosphoinositide 3-kinase regulatory subunit 4 (PIK3R4) [25], which plays a key role in autophagy activation.Te process of autophagy is regulated by more than 30 regulatory genes and multiple pathways.For example, the PI3K-AKT-mTORC1 pathway-induced autophagy was observed in isolated mouse fbroblasts [26].Te kinase ULK1 can activate autophagy by phosphorylating Beclin-1 that binds to ligands [27].Phosphatidylinositol-3-kinase complex can regulate autophagy through MAPK8/JNK1 activation.Bcl2 inhibits autophagy by interacting with BECN1.LC3 is an autophagy-associated core protein that is required for autophagosome formation and maturation [28].LC3 is present in the cytoplasm as LC3I, and LC3II is formed through binding LC2I to phosphatidylethanolamine after autophagy, which specifcally binds to phagosomes (precursors of autophagosomes) and induces autophagy [29].Diferent autophagic pathways may difer under diferent cellular or survival conditions.Our experiments demonstrated that AMPK/mTOR pathway-related proteins were little changed in LPS-induced rat cardiomyocytes, while P62, LC3I/II, and Beclin-1 protein levels were signifcantly altered.Moreover, the expression of these proteins could be regulated by NaHS to ameliorate the myocardial damage.H 2 S attenuated sleep deprivationinduced cognitive impairment by reducing excessive autophagy of hippocampal SIRT 1 in WISTAR rats [30].H 2 S protected retinal pigment epithelial cells from oxidative stress-induced apoptosis and afected autophagy [31].In addition, H 2 S attenuated acute myocardial ischemic injury by modulating autophagy and infammatory response under oxidative stress [32].
Noncoding RNAs (ncRNAs) play a wide range of regulatory roles in various diseases such as cardiovascular disease and are considered major players in physiological and pathological processes [33][34][35].miR-133a-3p, a muscle-enriched miRNA, is highly expressed in the heart.Several studies have previously demonstrated that miR-133a-3p expression levels are signifcantly elevated in the circulation of patients with coronary artery disease, making it a promising blood biomarker for coronary artery disease [36,37].Chen et al. found that miR-133a expression was elevated in sepsis and could regulate sepsis-induced organ dysfunction and infammatory responses by targeting SIRT1 [38].However, our study found that miR-133a-3p expression was decreased in LPS-treated cardiomyocytes, while the expressions of autophagy-related proteins were altered where Beclin-1 and LC3I/II expressions were increased and P62 expression was decreased.After NaHS treatment in rat cardiomyocytes, miR-133a-3p expression was elevated.Moreover, autophagy-related proteins were also altered after NaHS treatment, with decreased Beclin-1 6 Journal of Toxicology and LC3I/II expressions and increased P62 expression.It may be related to diferent cell models, and apoptosisrelated studies also showed similar results that miR-133a-3p inhibited apoptosis by binding downstream to the 3′ UTR structural domain of CDIP1 to reduce transcriptional levels [32].Te simulant of miR-133a-3p promoted the phosphorylation of AKT in HUVEC or H9c2 cells [39].
In our experiments, we found that autophagy-associated proteins were altered in cardiomyocytes after treatment with NaHS but reversed after inhibition of miR-133a-3p expression, suggesting that NaHS regulated autophagy in LPS-stimulated cardiomyocytes through miR-133a-3p and thus protected the myocardium.Although LPS-stimulated cardiomyocytes showed decreased ATP content and energy supply, the AMPK level was not afected.In spite of no activation in the AMPK/mTOR-related autophagic pathway, other autophagy-related proteins could regulate autophagy levels in cardiomyocytes.In summary, miR-133a-3p expression was decreased in LPS-treated H9c2 cells and miR-133a-3p expression level was signifcantly upregulated after NaHS treatment, while the inhibition of miR-133a-3p expression reversed the altered expression of autophagy proteins.Te abovementioned results demonstrated that NaHS could upregulate the level of miR-133a-3p and alter the expression levels of autophagy proteins.Meanwhile, we found that the expression of mTOR and AMPK was not signifcantly changed.Terefore, it could be suggested that NaHS had a protective efect on myocardial injury by promoting miR-133a-3p expression, thus inhibiting autophagy and restoring cellular ATP levels in cardiomyocytes.Te present study provided a new direction for the mechanism and treatment related to sepsis-induced myocardial injury, and further studies on their direct targets will be explored in future experiments.

Figure 1 :
Figure 1: Te expression levels of ATP and miR-133a-3p.(a) Total cellular ATP levels were determined by using an ATP assay kit.Relative ATP level in each group.(b) We measured the expression level of miR-133a-3p by qPCR.* P ≤ 0.05 compared with the control group and # P ≤ 0.05 compared with the respective LPS-treated group.

Figure 2 :
Figure 2: QPCR assay and WB were used to detect the expression of related indicators.(a) AMPK and mTOR expressions were determined by qPCR.(b) Western blotting with densitometric analysis of AMPK, mTOR, Beclin-1, P62, and LC3I/II.* P ≤ 0.05 compared with the control group and # P ≤ 0.05 compared with the respective LPS-treated group.

Figure 3 :
Figure 3: Te expression levels of ATP and miR-133a-3p.(a) Total cellular ATP levels were determined using an ATP assay kit.Relative ATP levels in each group.(b) We measured the expression level of miR-133a-3p by qPCR.* P ≤ 0.05 compared with the control group, # P ≤ 0.05 compared with the respective LPS-treated group, and & P ≤ 0.05 compared with the LPS + NaHS group.

Figure 4 :
Figure 4: QPCR assay and WB were used to detect the expression of related indicators.(a) AMPK and mTOR expressions were determined by qPCR.(b) Western blotting with densitometric analysis of AMPK, mTOR, Beclin-1, P62, and LC3I/II.* P ≤ 0.05 compared with the control group, # P ≤ 0.05 compared with the respective LPS-treated group, and & P ≤ 0.05 compared with the LPS + NaHS group.