Tannic Acid Alleviates Lipopolysaccharide-Induced H9C2 Cell Apoptosis by Suppressing ROS-Mediated ER Stress

Background: Sepsis-induced myocardial dysfunction (SIMD), which is one of the features of multiple organ dysfunction in sepsis with extremely high mortality, is characterized by impaired myocardial compliance. To date, there are few effective treatment options to cure sepsis. Tannic acid (TA) is reportedly protective during sepsis. However, the underlying mechanisms by which TA protects against septic heart injury remains elusive. Methods: We investigated the potential effects and underlying mechanisms of TA in alleviating lipopolysaccharide (LPS)-induced H9C2 cardiomyocyte cell apoptosis. H9C2 cells were treated with LPS (15 μg/mL), TA (10 μM) and TA+LPS. Control cells were treated with media only. Apoptosis was measured using ow cytometry, RT-PCR, and Western blotting analysis. Additionally, laser confocal immunouorescence analysis detected the production of reactive oxygen species (ROS). Western blot and RT-PCR were employed to detect ER stress-associated functional proteins. Results: The results demonstrated that TA reduced the degree of LPS-induced H9C2 cells injury, including the inhibition of ROS production and endoplasmic reticulum (ER) stress-associated apoptosis. ER stressassociated functional proteins, including ATF6, PERK, IRE1, XBP1s, and CHOP were suppressed in response to TA treatment. Furthermore, the expression levels of ER stress-associated apoptotic proteins, including JNK, Bax, Cyt, Caspase3, Caspase12, and Caspase9 were reduced following treatment with TA. Additionally, the protective effects of TA on LPS-induced H9C2 cells were strengthened following treatment with the ROS inhibitor, N-Acetylcysteine (NAC), which demonstrated that ROS-mediated ER stress-associated apoptosis and TA decreased ROS-mediated ER stress-associated apoptosis. Conclusion: Our ndings demonstrated that the protective effects of TA against LPS-induced H9C2 cells apoptosis may be associated with the amelioration of ROS-mediated ER stress. These ndings may assist the development of potential novel therapeutic methods to inhibit the progression of myocardial cell injury. (TA alleviates LPS-induced H9C2 cell apoptosis.)


Introduction
Sepsis is a life-threatening multiple organ dysfunction syndrome (MODS) characterized by a disordered immune, endocrine, and metabolic response to infection [1,2]. Severe sepsis may result in signi cant morbidity and mortality [3][4][5][6]. Myocardial dysfunctions are common among patients with severe sepsis.
Cardiac dysfunction is a well-recognized feature of MODS [7,8]. Sepsis-induced myocardial dysfunction (SIMD) was identi ed as a sepsis-associated acute syndrome of cardiac dysfunction. Many factors, such as lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-α) and IL-1 are associated with SIMD [9,10]. Several mechanisms have been proposed to explain the pathophysiology of SIMD, including excessive generation of in ammatory cytokines, reactive oxygen species (ROS) or nitrogen radicals, as well as transcriptional and compromised metabolic changes [11][12][13][14]. Despite extensive studies, the relevant pathogenesis of SIMD has not yet been clearly de ned to date [13,14].
LPS is a component of the outer membrane of Gram-negative bacteremia, it has been regarded as the principal cause responsible for cardiac dysfunction in sepsis [15]. LPS associates with its receptor, tolllike receptor 4, to stimulate the production of in ammatory cytokines and simultaneously generate abundant ROS, which might directly disturb cardiac function [13,14]. ROS are by-products of normal cellular oxidative processes, and are generated in response to external stimuli such as in ammatory cytokines including LPS, chemotherapeutic drugs, and ionizing radiation [16,17]. Excessive ROS, which is considered to be a marker of oxidative stress, causes cell apoptosis [18]. Enhancing the cellular antioxidant capacity or ROS-scavenging activity may act to ameliorate the imbalance in a variety of pathologies and disease models [14,16]. Excessive ROS production can trigger endoplasmic reticulum (ER) stress, which is called ROS-mediated ER stress [19]. Endoplasmic reticulum stress (ERS) is one of the mechanisms contributing to ROS-mediated cell apoptosis [20], and ERS-associated cardiomyocyte apoptosis is a major contributor to myocardial injury [21]. The ER organelle is responsible for the folding of secretory and membrane proteins. In mammals, the ERS response is mediated by three known transmembrane sensor proteins in the endoplasmic reticulum, namely the inositol requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and protein kinase-like endoplasmic reticulum kinase (PERK). Numerous intracellular and extracellular stimuli can cause the accumulation of unfolded and misfolded proteins, resulting in the dissociation of glucose-regulated protein 78 (GRP78) from the three ER transmembrane protein sensors PERK, ATF6 and IRE1, and subsequently trigger the unfolded protein response (UPR) [22]. If the stimulus is serious and/or lengthy, it can activate ER stress-related C/EBPhomologous protein (CHOP), caspase-12, and/or JNK-dependent apoptotic signaling pathways [23,24].
Attenuation of ER stress with pharmacological or gene therapy strategies has been successful in reducing pathological features in various experimental models of in ammatory diseases, including sepsis [25,26]. Therefore, SIMD may be alleviated by the regulation of ROS and ERS. There is urgent need for innovative therapeutic options for the treatment of SIMD, which is essential for decreasing mortality from sepsis.
Tannic acid (TA), a speci c type of tannin, is water-soluble polyphenols with a C 76 H 52 O 46 chemical structure, and is a mixture of ve galloyl esters and one glucose [27]. Previous studies have indicated that TA possesses anticarcinogenic, antioxidant, antimutagenic, anti-in ammatory, anti brotic, and cardioprotective effects [28][29][30][31][32]. To date, there are no published scienti c reports on the effects of TA on LPS-induced myocardial injury or related ailments. Whether ERS regulates the inhibitory effects of TA during LPS-induced myocardial dysfunction remains unknown. Additionally, the speci c mechanism by which TA alleviates myocardial dysfunction urgently needs to be investigated. Therefore, in this study we aimed to identify the role of TA in LPS-induced cardiomyocytes injury, and to elucidate the underlying mechanisms using H9C2 cells.

Antibodies
Primary antibody directed against GAPDH (catalog number ab9485) was purchased from Abcam

Cell culture and treatment
The H9C2 cells were cultured in high-glucose DMEM supplemented containing 10% (v/v) FBS, 1.5 g/L NaHCO 3 and 1% penicillin-streptomycin (Life Technologies Corporation, Carlsbad, CA, USA) at 37 °C in a humidi ed atmosphere with 5% CO 2, and maintained in a logarithmic growth phase for all experiments.

CCK-8 assay
The H9C2 cells were seeded in 96-well plate at a density of 5000 cells/well, and treated with the following conditions: fresh culture medium alone (control), fresh culture medium with different concentrations (0-50 µM) of TA, and/or fresh culture medium with 15 μg/mL LPS for the indicated times. Cell viability was assessed using the cell counting kit, CCK-8, according to manufacturer's instructions. Brie y, after treatment, the CCK-8 solution (10 µL) was added to each well and the cultures were incubated at 37 °C in humidi ed 95% air and 5% CO 2 for 1 h. Absorbance was measured at 450 nm using a Microplate Reader

Measurement of cellular ROS by laser confocal microscopy
The cellular ROS Detection Assay Kit contains the uorogenic dye, 2,7-dichloro uorescin-diacetate (DCF-DA), that measures ROS activity within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a non-uorescent compound, which is later oxidized by ROS into 2', 7'dichloro uorescein (DCF). DCF is a highly uorescent compound which can be detected by uorescence spectroscopy with excitation / emission at 485 nm / 535 nm. The H9C2 cells were incubated with 2,7dichloro uorescein-diacetate (DCFH-DA, 10µM, Jian cheng, China) for 30 min at 37 °C in the dark. The cells were subsequently washed twice with PBS and observed under a confocal laser scanning microscope (Leica), and the images were captured. The mean uorescence intensity of DCF was analyzed.

Flow cytometric analysis of apoptosis
Fluorescein-conjugated annexin V (annexin V-FITC) was used to measure the apoptosis in H9C2 cells as described previously [33]. The necrotic cells were counter stained with propidium iodide (PI). Cells were plated in 60-mm dishes and exposed to different treatments once reaching 80-85% con uence. Detached and adherent cells were collected, washed twice with cold PBS, and then resuspended in the 1X binding buffer at a concentration of 1×10 6 cells/mL. The resuspended solution containing 1×10 5 cells (100 µL) was transferred to a 5 mL culture tube, to which 5 µL of FITC Annexin V and 5 µL PI were added, and gently vortexed, prior to 15 min incubation at 25 °C in the dark. Binding buffer 1X (400 µL) was added to each tube, and the percentages of apoptotic cells were analyzed by ow cytometry within 1 h.

RT-PCR
Real-time quantitative polymerase chain reaction (RT-PCR) analysis was performed as described previously [34]. The primer sequences are described in Table 1. Brie y, total RNA was extracted using Trizol (catalog number 15596026, Life technologies, California, USA), and then DNase was used to remove internal DNA contamination. Random primers were used in reverse transcription reactions to obtain rst-strand cDNA, and SYBR Green qPCR Master Mix (catalog number FP202, Tiangen, Beijing, China) was used to amplify the target fragments. GAPDH cDNA ampli cation was used as an internal control. The general reaction procedure included an initial denaturing step at 95 °C for 15 minutes followed by 37 cycles of denaturing at 95 °C for 10 sec, annealing at 57 °C for 30 sec, and extension at 72°C for 30 sec, with a plate read at 95 °C for 5 sec. Each experimental group was performed in triplicate. IBM SPSS (version 19) software was used to analyze differences in expression.

Western blotting
Western blotting was performed as describing previously [35]. Brie y, cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, and 1% NP-40 with a protease inhibitor cocktail and phosphatase inhibitor cocktail, The insoluble components were removed by centrifugation at 12,000 × g for 15 minutes at 4 °C. Subsequently, protein concentrations were measured using the BCA method. After denaturation, the total proteins were separated by 10% SDS-PAGE and transferred onto 0.2 µm or 0.45 µm polyvinylidene uoride membranes. After blocking with 5% (w/v) non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The membranes were subsequently washed three times with 1 × TBST, and further incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. After subsequent washes of the membrane, three times with 1 × TBST, antibody detection was performed using the Immobilon Western Chemiluminescent HRP Substrate (ECL, catalog number WBKLS0500, Merck Millipore, MA, USA) and the detection kit using a ChemiDoc™ XRS+ system (Bio-Rad, Hercules, California, USA). ImageJ (version 1.46) was used to compare the gray values of the target bands; GAPDH served as an internal control.

Statistical analysis
Data is presented as the mean ± standard error of the mean (SEM) from at three independent experiments. GraphPad Prism 8.0.2 (GraphPad Software Inc., La Jolla, CA) was used for statistical analysis. Statistical signi cance was determined by one-way ANOVA with Bonferroni correction for multiple comparisons. A P value <0.05 was considered statistically signi cant. The number of independent experiments performed is indicated in the gure legends.

Results
Effect of TA on LPS-induced cell viability of H9C2 cells.
Prior to investigating whether TA protected H9C2 cells from LPS-induced cell injury, the cytotoxicity of the extract was determined by culturing H9C2 cells until reaching 80-90% con uence in 96-well plates, and treating them with different dosages of TA (0, 5, 10, 20, 30,40 and 50 µM) for 24 h. Cell viability assessment, using the CCK-8 assay, appeared to reveal slightly increased cell viability at TA concentrations less than 10 µM; however, these increases were not statistically signi cant when compared to the control group (Fig. 1a). Following treatment of H9C2 cells with TA concentrations greater than 30 µM, the cell viability signi cantly decreased. Thus, to exclude cell toxicity, concentrations of 5, 10, and 20 µM were selected for subsequent experiments. To examine the effects of TA on LPS-induced cell injury, the viability of H9C2 cells pretreated with 0, 5, 10 and 20 µM TA, and then treated with 15 µg/mL LPS was determined. As shown in Fig. 1b, LPS treatment signi cantly diminished cell viability to 50.65 ± 0.5% compared to control. Interestingly, pretreatment with TA (5, 10 and 20 µM) prior to the LPS treatment signi cantly improved cell viability to 68.3 ± 0.8%, 84.6 ± 2.1% and 90.6 ± 3.3%, respectively, compared to control. Our ndings demonstrated that pretreatment with TA dose-dependently increased the cell viability diminished by the LPS treatment, and suggested that TA is capable of protecting H9C2 cells from LPSinduced cell injury.
TA alleviates LPS-induced apoptosis in H9C2 cells.
To further evaluate the effect of TA on LPS-induced apoptosis, H9C2 cells were treated either with LPS or a combination of TA and LPS. Cell apoptosis was measured by ow cytometry, and the expression of apoptosis-associated proteins in the H9C2 cells was measured by western blotting. As presented in Fig. 2a and 2b, the percentage of apoptotic cells signi cantly increased after LPS treatment. Whereas, pretreatment with TA signi cantly decreased the percentage of apoptotic cells induced by the LPS treatment. Consistent with the ow cytometric results, LPS increased the protein expression of apoptotic markers Bax/Bcl2, Cleaved Caspase3 and Cleaved Caspase9, compared to the control group. However, pretreatment with TA decreased the expression of these markers compared to treatment with LPS alone (Fig. 2c and 2d). Notably, the detected levels of all markers in the TA-only group were comparable to the control (medium) treatment. Our results suggested that TA alleviated LPS-induced H9C2 cells apoptosis.

TA attenuates ROS generation in LPS-induced H9C2 cells.
The level of ROS is a crucial index of oxidative stress. To further evaluate the effects of TA on LPSinduced oxidative stress, the level of intracellular ROS was monitored by detecting changes in DCF uorescence by laser confocal microscopy. As shown in Fig. 3a and 3b, LPS treatment signi cantly elevated ROS generation to level of the control group treated with cultural medium. However, the LPSmediated increase in DCF uorescence was signi cantly decreased with TA pretreatment. Notably, the ROS levels detected in the TA-only treated group was comparable to the control group treated with medium only, demonstrating the minimum cell toxicity of this chosen TA dose. Our results suggested that TA was capable of reducing intracellular ROS accumulation following LPS stimulation. The ability of TA to inhibit intracellular ROS production contributed to the e cacy at protecting the H9C2 cells against LPS-induced apoptosis.

TA alleviates LPS-induced ER stress in H9C2 cells.
To determine the effect of TA on ERstress in H9C2 cells induced by LPS, the expression of ER stressassociated proteins was examined. As presented in Fig. 4a-c, the expression of ER stressassociated proteins proapoptotic CHOP, ATF6, Phospho-PERK, PERK, Phospho-IRE1/IRE1 were signi cantly increased after exposure to LPS compared to the control group. Additionally, the expression of ER stressassociated proteins were markedly downregulated in the TA and LPS group compared to the group treated with LPS only. This result suggested that pretreatment with TA signi cantly downregulated the expression of ER stressassociated proteins, which alleviated LPS-induced ER stress. Based on this nding, it is feasible to hypothesize that the ER stressassociated proteins proapoptotic CHOP, ATF6, PERK and IRE1 maybe underlying players involved in alleviating LPS-induced H9C2 cell apoptosis by TA.
TA attenuated LPS-induced apoptosis by suppressing ROS-mediated ER stress in H9C2 cells.
We further examined the protective effects of TA on H9C2 cells and explored the relationship between ROS and ER stress-associated apoptosis in LPS-induced H9C2 cells. To determine whether LPS-induced ROS modulated ER stress in H9C2 cells we used the ROS scavenger, NAC, to evaluate the relationships between ROS, ERS, and apoptosis. As presented in Fig. 5a-c and 5i-k, H9C2 cells were pretreated with NAC 1 mM or TA 10 µM, and then co-treated with LPS 15 µg/mL. The protein expression levels of ER stressassociated proteins (CHOP, ATF6, Phospho-PERK, PERK, Phospho-IRE1, and IRE1) and apoptosisassociated proteins (p-JNK, JNK, Cleaved Caspase3, Bax/Bcl-2, and Cleaved Caspase9) were detected by western blotting. The detected mRNA expression levels of ER stressassociated genes (Chop, Atf6, Perk, Ire1, and Xbp1s) and apoptosis-associated genes (Bax, Caspase3, Caspase12, and Cyt) are described in Fig. 5d-h and 5 l-o. LPS caused a signi cant increase in the expression of ER stressassociated proteins and apoptosis-associated proteins; however, these protein levels were markedly decreased after TA or NAC pretreatment. Moreover, The expression of ER stressassociated proteins and apoptosis-associated proteins couldn't be inhibited by TA in the presence of ROS inhibitors such as NAC. The levels of all markers detected in the NAC-only group were similar to that observed in the medium control treatment group. Our results indicated that ER stress activation and apoptosis activation were inhibited by TA in a ROS-dependent manner. ER stress is considered as one of the mechanisms contributing to ROS-mediated cell apoptosis [20]. Our results also demonstrated that LPS-induced ROS modulated the ER stress in H9C2 cells, suggesting that the ROS-mediated ER stress pathway may be one of the important mechanistic pathways modulating apoptosis in LPS-induced H9C2 cells. Taken together, TA prevented LPS-induced ROS-mediated ER stress, which may be responsible for the TA-regulated decrease in H9C2 cells apoptosis.

Discussion
The major ndings of this study are that (a) the use of appropriate TA concentrations promoted the increase of cell viability in LPS-induced H9C2 cells, (b) TA attenuated ROS, ER stress, and apoptosis in LPS-induced H9C2 cells, (c) suppression of ROS-mediated ER stress-related apoptosis in LPS-induced H9C2 cells was strongly involved in the protective effects of TA. Collectively, our data demonstrated that TA protected against LPS-induced H9C2 cells apoptosis for the rst time. Additionally, we observed that TA reduced the overproduction of ROS, as well as the level of ER stress.
Sepsis is a serious disease with extremely high mortality, similar to that of acute myocardial infarction [36,37]. SIMD was de ned as asepsis-associated acute syndrome of cardiac dysfunction. As there is still no effective treatment strategy for SIMD, there is an urgent need to explore the effective remedies and innovative treatment options to combat SIMD. LPS, the major compontent of bacterial outer membrane, is considered the principal cause responsible for multiorgan failure in sepsis, including myocardial depression [38,39]. LPS-induced H9C2 cells are often used in the study of septic cardiomyopathy. Several mechanisms have been proposed to explain the pathophysiology of SIMD, including the excessive generation of in ammatory cytokines [40], ROS [41], cell death (necrosis and apoptosis) [42], impairment of nitricoxide [43], over-activation of ERS [26] and transcriptional and comprised metabolic changes [44]. Therefore, anti-in ammatory agents, anti-apoptosis agents, and anti-oxidants could be promising drugs to ameliorate SIMD.
TA is a natural polyphenol which has been used to improve human health [45]. TA is reported to alleviate acute lung injury [46], cardiac hypertrophy [47], acute doxorubicin-induced cardiotoxicity [48], and isoproterenol-induced myocardial injury [32]. However, there are no reports on the effects of TA on LPSinduced cardiomyocytes injury. This study was rstly performed to evaluate the possible mechanisms of action underlying tannic acid-mediated prevention of cardiomyocyte H9C2 cell injury. Our study showed that administration of TA at high doses (40 and 50 µM) induced cell injury (Fig. 1a), whereas doses of TA lower than 20 µM (including 20 µM) were nontoxic to H9C2 cells. When investigating the protective effect of TA against LPS-induced cell injury, using the viability CCK8 assay, we observed the capacity of TA to protect H9C2 cells from LPS-induced cell injury in a dose-dependent manner (Fig. 1b).
ROS is well-known to trigger apoptosis through activation of essential stress signaling molecules, including c-Jun N-terminal kinase (JNK) [49,50]. Moreover, the mitochondrion is a cause of ROS. Mitochondrial dysfunction is reportedly associated with cytochrome c release and the loss of mitochondrial membrane potential, ultimately resulting in increased apoptosis [51]. ROS is a key initiation factor for oxidative stress; excessive ROS increases in the body can induce oxidative stress [52,53]. Many studies have shown that oxidative stress plays an important role in sepsis pathogenesis [11,54]. In our study, we also found that ROS was increased in LPS-induced H9C2 cells (Fig. 3). However, TA was able to signi cantly inhibit ROS and apoptosis in the LPS-induced H9C2 cells (Fig. 2-3). This rstly con rmed the potential antioxidant and anti-apoptotic function of TA in LPS-induced H9C2 cells.
In addition to the dangerous and rapid reactivity against cellular components, ROS is recognized as potential signaling molecules for initiating various cellular events [55]. Many studies have suggested that ER stress is often induced by increased ROS production within the myocardium [56,57], the overexpression of ROS may further result in the dysregulation of the ER, complex UPR signaling pathway and cell apoptosis. In our study, we also determined the close relationship between ROS and ER stress. We found that ROS-mediated ER stress-related apoptosis in LPS-induced H9C2 cells (Fig. 5).
ER stress is mediated by three known transmembrane sensor proteins, namely inositol requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and protein kinase-like endoplasmic reticulum kinase (PERK) [58]. ER stress-related CHOP, Caspase12, and /or JNK-dependent apoptotic signaling pathways can be activated by a severe stimulus, which in turn affects the expression of Bax/Bcl-2 and the activation of Caspase3 [24,59]. High expression of CHOP upregulates the cytoplasmic pro-apoptotic protein Bax, and the release of Cyt from the mitochondria, leading to the activation of Caspase3, Caspase9 and the induction of cell apoptosis [60]. In our study, we also observed that ER stress was triggered after LPS administration, eventually resulting in H9C2 cell injury. Additionally, we found that TA was able to signi cantly inhibit ER stress in the LPS-induced H9C2 cells (Fig. 4). There was no research on effect of TA on the link of ROS and ER stress-related apoptosis in LPS-induced H9C2 cells. Therefore, further investigation is needed to determine whether ER stress plays an important role in ROS-scavenging by TA to reduce cardiomyocyte H9C2 cell injury. NAC, a well-known ROS scavenger, was utilized as a positive control to treat H9C2 cells. In our experiments, LPS-induced H9C2 cells were preincubated with TA and /or NAC. Our data demonstrated that inhibition of ROS signi cantly suppressed the expression of ER stress-associated functional proteins induced by LPS. However, the expression of ER stressassociated proteins couldn't be suppressed by TA in the presence of NAC (ROS inhibitors) in LPS-induced H9C2 cells ( Fig. 5a-h). We also suggested that the inhibition of ROS signi cantly suppressed ER stress-associated apoptotic proteins, including JNK, Bax, Cyt, Caspase3, Caspase12 and Caspase9. Moreover, the expression of ER stress-associated apoptotic proteins couldn't be suppressed by TA in the presence of NAC in LPS-induced H9C2 cells (Fig. 5i-o). Our results demonstrated that ER stress activation and apoptosis activation were inhibited by TA partly in a ROS-dependent manner.
In conclusion, our study rstly demonstrated that TA may protect LPS-induced H9C2 cells against damage via the suppression of ROS-mediated ER stress-associated apoptosis, providing a comprehensive understanding of the mechanisms underlying the cardioprotective role of TA. Based on this, TA may be targeted as an e cient therapeutic drug for the treatment of SIMD to improve heart function. Although our study suggests that the ROS-mediated ER stress-associated apoptosis exerts important functions in LPS-induced H9C2 cell injury model, there may be some differences in an in vivo context. H9C2 cells were originally derived from embryonic cardiac tissue; however, in vivo cardiac myocytes have more complex functions. To better understand the mechanisms involved in cardiac injury, additional studies are needed in the future to evaluate these ndings, and to identify more speci c mechanisms in vivo.
Abbreviations TA, tannic acid; LPS, lipopolysaccharide; SIMD, sepsis-induced myocardial dysfunction; ROS, reactive oxygen species; ERS, endoplasmic reticulum stress; UPR, unfolded protein response; IRE1, inositol requiring enzyme 1; ATF6, activating transcription factor 6; PERK, protein kinase-like endoplasmic reticulum kinase; XBP1s, spliced X box-binding protein 1; CYT, cytochrome c; CHOP, C/EBP-homologous protein; JNK, c-Jun N-terminal kinase; NAC, N-Acetylcysteine Declarations Tables   Table 1 Primers used for real-time qPCR analyses in this study Target genes Primer sequences   TA attenuates ROS production in LPS-induced H9C2 cells. Data represents ROS uorescence intensity using the DCFH-DA assay. a Laser confocal microscopy measurement of cellular ROS production by H9C2 cells treated with LPS, TA and TA+LPS (scale bar=100 μm) b Quantitative analysis of the mean uorescence intensity following treatment with LPS, TA and TA+LPS. Data is presented as mean ± SEM of at least three independent experiments. Statistical signi cance is de ned as *p < 0.05 and **p < 0.01 and ***p < 0.001 vs. cont; #p < 0.05 and ##p < 0.01 vs. LPS alone group. ROS, reactive oxygen species; TA, tannic acid; LPS, lipopolysaccharide; cont, control.