Trans-sodium crocetinate suppresses apoptotic and oxidative response following myoglobin-induced cytotoxicity in HEK-2 cells

doi:10.21203/rs.3.rs-3140940/v1

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Trans-sodium crocetinate suppresses apoptotic and oxidative response following myoglobin-induced cytotoxicity in HEK-2 cells

https://doi.org/10.21203/rs.3.rs-3140940/v1

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Purpose: Rhabdomyolysis (RM) is a serious fatal syndrome. The RM leads to acute kidney injury (AKI) as a fatal complication. The belief is that the RM-induced AKI is triggered by myoglobin (MB). MB activates oxidative and apoptotic pathways.Trans-sodium crocetinate (TSC) isobtained from saffron and it has antioxidant andrenoprotective effects. This research was designed to assess the mechanisms of MB-induced cytotoxicity in HEK-2 cells (human embryonic kidney cells) as well as the possible effects of TSC against MB-induced cytotoxicity.

Methods: HEK-2 cells were exposed to diverse concentrations of TSC (2.5, 5, 10, 20, 40, 80, and 100 µM) for 24 h. Then, MB (9 mg/ml) was added to the cells. After 24 h, cell viability was measured through MTT, and The values of ROS generation were calculated using DCFH-DA assay. Besides, using western blot autophagy and apoptosis markers in cells were assessed.

Results: MB decreased viability and increased ROS levels in HEK-2 cells. Furthermore, MB enhanced the apoptosis markers (cleaved caspase-3 and Bax/Bcl-2 ratio) and autophagy markers (LC3II/I ratio and Beclin-1) in HEK-2 cells. However, pretreatment of HEK-2 cells with TSC for 24 h reduced the cytotoxicity and ROS production caused by MB. Additionally, TSC pretreatment condensed the levels of autophagy and apoptosis markers in response to MB cytotoxicity.

Conclusion:TSC has a positive effect in preventing MB-induced cytotoxicity in HEK-2 cells by increasing antioxidant activity and regulation of apoptotic and autophagy signaling pathways.

Apoptosis

Autophagy

Rhabdomyolysis

Acute kidney injury

Trans-sodium crocetinate

Myoglobin

Rhabdomyolysis (RM) is a medical disorder described through damage to skeletal muscle fibers with the distribution of their whole contents to the bloodstream. Myoglobin (MB), lactate dehydrogenase and creatine phosphokinase are the greatest important substances released into the blood circulation (Zutt et al. 2014). Acute kidney injury (AKI) is an important and known complication of RM. MB induces intrarenal vasoconstriction, tubular obstruction and direct tubule injury which are mainly involved in RM-induced AKI (Lafrance and Miller 2010).

RM-induced AKI is triggered by MB which induces renal dysfunction (Zhou et al. 2012). MB is an iron and oxygen-binding protein (Zager 1996). MB significantly releases from myocytes when skeletal muscle fibers distribute and RM occurs. The clinical study demonstrated that renal uptake of MB is mediated via endocytic receptors (Luo et al. 2016). MB is the most significant toxic factor that causes renal cell injury through direct and indirect mechanisms (Wang et al. 2011).

MB is promoting reactive oxygen species (ROS) generation and redox-active iron generation, apoptosis and inflammation (Zhou et al. 2012). The permeability of the mitochondrial membrane is altered by MB, which induces the secretion of cytochrome c, caspase-3 activation, and renal tubular cell apoptosis. The former investigation suggested that, besides apoptosis, other forms of cell death may be attributed to MB-induced cytotoxicity (Zager 1996). Taken together, results confirmed that MB-induced oxidative damage is a significant cause of renal cell apoptosis in RM-induced AKI (Chen et al. 2017).

In vivo models of AKI induced by glycerol in rats and in vitro models of MB-induced cytotoxicity in HEK-2 cells (human embryonic kidney cells) are commonly used to study RM-induced AKI. These models allow researchers to investigate the underlying mechanisms of RM-induced AKI and to test potential therapeutic interventions (Chen et al. 2017). In vitro studies are conducted to investigate the direct cytotoxic effects of MB on renal cells, as there are no intervening factors such as hypoxia after vasoconstriction which occur in vivo (Zhou et al. 2012).

The occurrence of RM-induced AKI has enhanced over the prior decade, so, in recent years, research on useful therapies to prevent RM-induced AKI has attracted much attention (Zutt et al. 2014). Herbal therapy plays a significant and major role in the landmark of healthcare. This could be due to cultural acceptability, safety, potency, and fewer side effects (Razavi and Hosseinzadeh 2017). Saffron is a traditional and well-known herb, that belongs to the Iridaceae family (Omidkhoda and Hosseinzadeh 2022) and has a long history in traditional medicine (Bukhari et al. 2018). Crocetin is an ingredient of saffron (Khoshandam et al. 2022), which exhibits various pharmacological attributes such as hepatoprotective (Chen et al. 2016), neuroprotective (Ahmad et al. 2005), cardioprotective (Ghorbanzadeh et al. 2017) and anti-atherosclerosis (Yu et al. 2021). The renoprotection property of crocetin, can be mediated through antioxidant (Rajabian et al. 2023), anti-inflammatory (Hashemi and Hosseinzadeh 2019), anti-apoptotic (Moshiri et al. 2015), and autophagy regulation (Nassiri-Asl and Hosseinzadeh 2020) mechanisms. Crocetin normalized intracellular Ca²⁺ homeostasis, scavenged intracellular ROS, and raised antioxidant protection in bovine aortic endothelial cells (Xiang et al. 2006). It prevented inflammatory cytokines production, elevated anti-inflammatory cytokine in plasma and inhibited nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in APPsw transgenic mice because crocetin has a pseudo-nonsteroidal anti-inflammatory drug (NSAID) character (Zhang et al. 2018). Crocetin diminished extracellular signal‑regulated protein kinase (ERK1/2), phosphoinositide 3-kinases (PI3K) /AKT and p38 expression in KYSE-150 cells and consequently inhibited mitochondrial-mediated apoptosis (Li et al. 2019). Also, in HEK-2 cells, crocetin repaired the antioxidant system and prevented ROS production. Likewise, it prevented the ROS release from mitochondria into the cytoplasm through the steadiness of the mitochondrial membrane potential (Pradhan et al. 2019). Numerous lines of documents reported the advantageous effects of crocetin as a potent antioxidant in the protection of kidney diseases (Bandegi et al. 2014). Therefore, crocetin can be a useful remedy for MB-induced cytotoxicity.

Trans-sodium crocetinate (TSC) is a popular trans-carotenoid salt, which is obtained from crocetin (Lapchak 2010). TSC increases the oxygen transfer from erythrocytes to tissues and oxygen diffusivity and decreases plasma resistance (Rameshrad et al. 2018). So it has been developed to enhance the delivery of oxygen (O₂) to damaged and hypoxic tissues (Gainer et al. 2005). Additionally, TSC can enhance the effects of crocetin by improving its bioavailability (Wang et al. 2014).

Thus, the present investigation was conducted to assess the effect of TSC on MB-induced cytotoxicity in HEK − 2 cells with special attention to cellular signaling pathways including, oxidative stress, apoptosis and autophagy.

MB from equine skeletal muscle was obtained from Sigma (Munich, Germany). TSC and vitamin C (Vit C) were bought from Tinab Shimi (Mashhad, Iran). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) high glucose were prepared from Gibco (New York, USA). Fluorescent probe 2, 7-dichlorofluorescein diacetate (DCF-DA) and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) were bought from Sigma (Munich, Germany). The polyvinylidene fluoride (PVDF) membrane was prepared from Bio-Rad (CA, USA).

Cell culture studies

In the present research, HEK-2 cells were purchased from the Pasteur Institute (Tehran, Iran). In summary, HEK-2 cells were cultured in DMEM complemented with streptomycin (100 µg/ml), penicillin (100 U/ml), and heat-inactivated FBS (10% v/v). HEK-2 cells were preserved in a humidified atmosphere (90%) at 37°C containing 5% CO2 in the air (Lee et al. 2009).

MB-induced cytotoxicity

MB was prepared in the reduced condition by dissolving 356 mg MB and 32.5 mg Vit C in DMEM (100 ml). The solution was blended and filtered via a 0.22-µm filter (Chen et al. 2017). In the MB-induced cytotoxicity group, HEK-2 cells were cultured for 24 h and then incubated for another 24 h with DMEM containing reduced MB. In the crocetin intervention group, at first, cells were pre-traded with diverse concentrations of TSC (2.5, 5, 10, 20, 40, 80, and 100 µM) for 24 h and then exposed to MB.

Cell viability assay

The viability of HEK-2 cells was surveyed via the MTT test in 96-well microplates (2500 cells/well). To assess the cytotoxic effect of MB on cells, the attached HEK-2 cells were incubated in DMEM at 37°C with several concentrations of MB (0–15 mg/ml) for 24 h, and the cell viability was determined by MTT test. The GraphPad Prism 8 statistical software was used to evaluate the concentration of MB that leads to 50% of cell death (IC₅₀). To investigate the cytoprotective effect of TSC, HEK-2 cells were incubated at 37°C in DMEM for 24 h. Afterward, various nontoxic concentrations of TSC (2.5, 5, 10, 20, 40, 80, and 100 µM) were added and the plate was incubated for 24 h. Then, MB at a concentration of 9 mg/ml was added and incubated for the next 24 h. Finally, the cells were treated with MTT solution (0.5 mg/ml), and incubated at 37°C. After 2 h the medium was eliminated and 100 µl dimethylsulfoxide (DMSO) was added individually to each well and have been shaken in the dark and at room temperature for up to 15 min for solving purple formazan crystals. Using an ELISA reader (Start Fax-2100, UK) the absorbance was measured at the wavelength of 545 and 630 nm.

Measurement of intracellular ROS generation

The accumulation of intracellular ROS was determined by the fluorometric technique (DCFH-DA assay) (Li et al. 2008). HEK-2 cells (2500 cells/well) were cultured in plates (96-well) and incubated for 24 h. The cells were pre-treated with different concentrations of TSC (1, 2.5, 5, 10, 20, 40, 80, and 100 µM) for 24 h. Afterward, MB (9 mg/ml) was added and incubated for another 24 h. Following incubation, cells were washed with PBS 2 times and then incubated at 37°C with DCFH-DA (10 µM) for 30 minutes. The fluorescence intensity of DCF was assessed at 485 nm (excitation wavelength) and 528 nm (emission wavelength).

Western blot analysis

The expression of autophagy proteins and apoptotic pathway proteins were determined through the western blot technique in HEK-2 cells. Following the exposure of HEK-2 cells to TSC (40 µM) and MB (9 mg/ml), the cell pellets were collected and homogenized in the lysis buffer containing 50 mM Tris-HCl (pH: 7.4), 2 mM EDTA, complete protease inhibitor cocktail, 1 mM sodium orthovanadate (Na₃VO₄), 10 mM NaF, 2 mM EGTA, 10 mM beta-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.2% W/V sodium deoxycholate. The homogenized samples were sonicated for 40 seconds on ice via a probe sonicator (UP100H, Germany) to destroy the cell membrane and then centrifuged at 10,000 rpm for 10 minutes, at 4°C. The Bradford protein assay was done to determine the concentrations of protein in the supernatants. Homogenate extracts were blended with loading buffer (9:1 v/v; glycerol 20% v/v, Tris-base 100 mM, SDS 20% w/v, 2-ME 10% v/v, and bromophenol blue 0.2% w/v) and subsequently incubated in boiling water for 5 minutes. The samples were then aliquoted and kept at − 80°C. Supernatants with equal contents of total proteins were loaded and electrophoresed in 12% SDS-PAGE gels and transferred to PVDF membranes. Each membrane was individually blocked for 2 h using Tris-buffered saline with tween 20% (TBST) containing 5% dry skim milk at room temperature. Following that, the membranes were incubated on a rocker with primary antibodies (Cell Signaling) at 1:1000 dilutions, against Beclin-1, LC3 II/I, cleaved caspase-3, Bcl-2, Bax, and mouse beta-actin overnight at 4°C. Afterwards the membranes were washed and incubated with anti-rabbit IgG labeled with horseradish peroxidase or anti-mouse IgG labeled with the enzyme at 1:3000 dilutions with gentle shaking, for 2 h at room temperature. Lastly, the optical densities of the bands were quantified by Alliance 4.7 Geldoc (UK). UVtec software (UK) was done for the densitometric analysis of the protein bands. In each membrane, protein bands were normalized relative to their β-actin protein as control (Mehri et al. 2012).

Statistical analysis

The data are presented as mean ± standard deviation (SD). One-way ANOVA for statistical analyses and then the Tukey-Kramer post-test to compare means were used (Prism 8.0, GraphPad Software Inc., CA, USA). Differences were considered statistically significant when P was less than 0.05. The IC₅₀ values were evaluated using Prism 8.0, GraphPad Software Inc., CA, USA.

Effect of MB on cell viability in HEK-2 cells

Using the MTT test, the cytotoxicity of MB in HEK-2 cells was assessed. In comparison to the control group, HEK-2 cell viability exposed to MB for 24 h was diminished in a concentration-dependent manner. The IC₅₀ value of MB following 24 h exposure was 9.19 ± 0.02 mg/ml (Fig. 1).

Effect of TSC on cell viability in HEK-2 cells

TSC in the diverse concentrations )0-100 µM (after 48 h exposure, had no significant toxic effect on HEK-2 cells in comparison to the control group (Fig. 2).

Effect of TSC on MB-induced cytotoxicity in HEK-2 Cells

The viability of HEK-2 cells exposed to MB (9 mg/ml) for 24 h was meaningfully reduced compared to the control group (P < 0.001). However, pretreatment of HEK-2 cells with TSC (2.5, 5, 10, 20, 40, 80, and 100 µM) for 24 h meaningfully diminished cytotoxicity in comparison to the MB group (P < 0.001) (Fig. 3). Vit C had no significant toxic effect on HEK-2 cells.

Effect of TSC on MB-induced ROS generation in HEK-2 cells

Treatment of HEK-2 cells with MB at a concentration of 9 mg/ml for 24 h significantly elevated intracellular ROS content in comparison to the control group (P < 0.001). However, pretreatment with TSC at different concentrations (2.5, 5, 10, 20, 40, 80, and 100 µM) suppressed the production of ROS levels compared to the MB group (P < 0.001). Vit C did not enhance the levels of ROS in contrast with the control group (Fig. 4).

Effect of MB and TSC on apoptotic factor in HEK-2 Cells

To measure the expression of an apoptotic factor in HEK-2 cells western blotting assay was done. According to Figs. 5 and 6, exposure of HEK-2 cells to MB (9 mg/ml) for 24 h meaningfully increased cleaved caspase-3 (P < 0.001 vs control) and the Bax/Bcl-2 ratio (P < 0.001 vs control).

On the other hand, in cells exposed to MB, pretreatment with TSC (40 µM, for 24 h) decreased cleaved caspase-3 (P < 0.05) and the Bax/Bcl-2 ratio (P < 0.001) in comparison to the MB group

Effect of MB and TSC on autophagy proteins in HEK-2 cells

Western blotting was done for evaluating the effect of MB and TSC on the expression of key factors of autophagy downstream. Following exposure of HEK-2 cells to MB (9 mg/ml) for 24 h, LC3-II/I ratio (P < 0.01) and Beclin-1 (P < 0.05) were meaningfully enhanced compared with the control group. Pre-incubation of HEK-2 cells with TSC (40 µM, for 24 h) resulted in a reduction in the LC3-II/I ratio (P < 0.01) and Beclin-1 level (P < 0.05) in comparison with the MB group (Figs. 7 and 8).

The present research assessed the protective effect of TSC on MB-induced cytotoxicity through numerous signaling pathways in HEK-2 cells. Our results revealed that the exposure of HEK-2 cells to MB for 24 h decreased viability and increased ROS production. Furthermore, MB enhanced the apoptosis and autophagy markers in cells. However, pretreatment of HEK-2 cells with TSC for 24 h reduced the cytotoxicity and ROS production caused by MB. Additionally, TSC pretreatment diminished the levels of autophagy and apoptosis markers in response to MB cytotoxicity.

RM is a term used to describe the breakdown of skeletal muscle. RM leads to the release of cellular contents including enzymes, MB, and electrolytes into the extracellular fluid (Reis et al. 2019). MB is an oxygen-binding hemoprotein that releases from myocytes, and it is the main toxic factor that can result in renal injury. The development of oxidative stress is the main reason for RM-induced AKI (Chen et al. 2017). Recently, the incidence of RM has risen. Hence, the management of AKI as a deadly complication of RM is very necessary (Zutt et al. 2014). Herbal medicine such as saffron can affect in treatment of RM-induced AKI (Rameshrad et al. 2018).

In vivo model to study RM-induced AKI is focused on glycerol-induced AKI (Chen et al. 2017). On the other hand, in vitro studies are established to assess the direct cytotoxic effects of MB on HEK-2 cells (Zhou et al. 2012). In vitro investigations have shown that cell viability was reduced in HEK-2 cells that were exposed to 200 mM MB for 24 h (Zhou et al. 2012). Furthermore, Chen et al, observed that MB reduced cell viability in HEK-2 cells (Chen et al. 2017). In the present study, MB at concentrations of 0 − 15 mg/ml for 24 h concentration-dependently reduced the HEK-2 cells' viability and the IC₅₀ value of MB was 9.19 ± 0.02 mg/ml.

Exposure to MB causes oxidative stress-mediated cellular damage, which can reduce antioxidant enzyme activity and antioxidant levels. Fenton reaction produces hydroxyl radicals and induces significant damage in renal cells (Holt and Moore 2001). In our investigation, following exposure of HEK-2 cells to MB (9 mg/ml) for 24 h, ROS generation was enhanced. Our findings support prior research indicating oxidative damage is a primary cause of cell death in MB-induced cytotoxic HEK-2 cells (Chen et al. 2017).

TSC is an effective antioxidant that reduces intracellular ROS (Stennett et al. 2007). It enhances the activity of the internal antioxidant enzymes and clears free radicals (Pradhan et al. 2019). In a previous study, pre-treatment of bovine aortic endothelial cells with crocetin (1 M) for 12 h revealed that it reduced ROS formation (Xiang et al. 2006). Additionally, crocetin (0.1, 0.5, and 1 µM, for 24 h) was found to enhance antioxidant enzymes, leading to the detoxification of free radicals in cyclosporine A-mediated cytotoxic in HEK-2 cells (Pradhan et al. 2019). According to previous research projects, we realize that oxidative stress has a basic and important figure in MB toxicity in HEK-2 cells. Overall, our results confirmed the previous studies that the pretreatment of HEK-2 cells with TSC (2.5, 5, 10, 20, 40, 80, and 100 µM) for 24 h markedly increased cell viability and reduced the formation of ROS.

The evidence suggested that RM-induced AKI occurs through several molecular signal pathways. Among these, more evidence exhibited that apoptosis and oxidative stress were most implicated in MB-induced cytotoxicity (Panizo et al. 2015). In one of these studies, MB (200 Mm) elevated protein expression of caspase-9 and cytochrome C but not caspase-8 after 24 h exposure. These findings indicated that the intrinsic pathway plays a main figure in MB-induced HEK-2 cell apoptosis (Zhou et al. 2012). In agreement with prior reports, our data displayed that MB triggered the overexpression of Bax and cleaved caspase-3, along with reduced Bcl-2 levels in HEK-2 cells after 24 h.

Many studies have shown the protective figure of crocetin against cellular apoptosis. Crocetin is increased antioxidant activity and is adjusted intracellular homeostasis in vascular endothelial cells (20). Additionally, crocetin has been found to increase intrinsic mitochondrial toleration against apoptotic motives in cyclosporine A-mediated toxicity in HEK-2 cells (Pradhan et al. 2019) and fixes the potential of the mitochondrial membrane against radiation-induced damage in intestinal epithelial cells (Zhang et al. 2020).

Crocetin was found to protect against arsenic trioxide-induced kidney damage by diminishing apoptosis in rats, according to Liu et al. Crocetin inhibited caspase-3 and Bax while increasing the level of Bcl-2 (Liu et al. 2020). Furthermore, several studies have displayed that TSC can modulate the expression of apoptotic proteins (Hashemi and Hosseinzadeh 2019). In one of these investigations, TSC (1 and 5 M) for 48 h lowered cleaved caspase-3 and the level of Bax/Bcl-2 ratio in HEK-2 cells exposed to contrast media (Rajabian et al. 2023). In another investigation, myocardial ischemia/reperfusion injury increased apoptosis in the heart, despite TSC treatment showing cardioprotective effects via anti-apoptosis actions (Chang et al. 2019). Consistent with previous results, in this study, TSC (40 µM, for 24 h) pre-incubation of HEK-2 cells reduced cleaved caspase-3 and the level of Bax/Bcl-2 ratio.

Autophagy is a known self-digesting reply to lysosome-mediated degradation of injured cellular organelles or proteins and recycles nutrients for cell survivorship (Yang et al. 2013). According to research, in AKI, the fast and main function of autophagy has a protective figure in renal cells (Bork et al. 2020). After MB exposure, proteins and organelles are injured, and the lysosomes are increased, as a result, autophagy is stimulated and may ameliorate renal cell survivorship (Chen et al. 2017).

Research exhibited that meglumine diatrizoate (100 mg/ml) elevated the interpretation markers of autophagy in HEK-2 cells (Jiang et al. 2020). Similarly, in an in vitro study the LC3 II/LC3 I ratio was enhanced after HEK-2 cells were exposed to 200 mg/ml of iohexol for 6 h (Liu et al. 2017). Our findings were in line with the former research that MB (9 mg/ml) for 24 h enhanced the LC3 II/I ratio and Beclin-1 expression in HEK-2 cells.

Crocetin demonstrated beneficial effects in breast cancer cells (MCF-7) by regulating the autophagy markers (reduced Beclin-1 level) (15). Also, according to the research by Rajabian et al, TSC diminished autophagy marker levels (LC3II/I ratio and Beclin-1 level) in HEK-2 cells exposed to contrast media (Rajabian et al. 2023). Our finding suggested that TSC pre-treatment (40 µM, for 24 h) reduced the Beclin-1 level and the LC3 II/I ratio in HEK-2 cells, which supported previous studies linked to TSC-regulated autophagy.

In conclusion, our study displays that TSC can protect HEK-2 cells from MB-induced cytotoxicity. MB causes cytotoxicity in HEK-2 cells by increasing oxidative stress. Furthermore, MB increases apoptosis (as evidenced by changes in Bax/Bcl-2 ratio and cleaved caspase-3 level) and autophagy markers (as evidenced by changes in Beclin-1 level and LC3 II/I ratio). TSC protects against MB-induced cytotoxicity via anti-oxidant characteristics, autophagy pathway regulation, and anti-apoptotic properties (as depicted in Fig. 9).

RM: Rhabdomyolysis; AKI: Acute kidney injury; MB: Myoglobin; TSC: Trans-sodium crocetinate; HEK-2 cells: Human embryonic kidney cells: Bax: Bcl-2 associated X; Bcl-2: B-cell lymphoma 2; DMSO: Dimethyl sulfoxide; DTNB: 5,5-dithio-bis-2-nitrobenzoic acid; EDTA: Ethylenediaminetetraacetic acid; EGTA: Ethylene glycol tetraacetic acid; MTT: Thiazolyl blue; PMSF: Phenylmethanesulfonyl fluoride; PVDF: Polyvinylidene difluoride; ROS: Reactive oxygen species; SDS: Sodium dodecyl sulfate; TBA: 2-thiobarbituric acid; TBST: Tris-buffered saline tween; TCA: Trichloroacetic acid; NSAID: Pseudo-nonsteroidal anti-inflammatory drug; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; ERK: Extracellular signal‑regulated protein kinase; PI3K: Phosphoinositide 3-kinases; Vit C: Vitamin C; 2-ME: 2-mercaptoethanol; DMEM: Dulbecco’s modified Eagle’s medium; FBS:Fetal bovine serum; DCF-DA: Fluorescent probe 2, 7-dichlorofluorescein diacetate; SD: Standard deviation; MB IC₅₀: The concentration of MB that leads to 50% of cell death

Data availability statements

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

Not applicable

Consent for publication

All authors have agreed to the contents and approved the final version for publication.

Competing interests

The authors declare that they have no conflicts of interest.

Funding

This research was supported by the Vice-Chancellor of Research, at Mashhad University of Medical Sciences (Grant No: 991907).

Authors' contributions

HH and SM were supervisors, designed the work, revised it critically for important intellectual content, and approved the version to be published. TAF did the experiment, analyzed the data and wrote the manuscript, MGR and FR helped in doing the research. All authors have read and approved the paper.

Acknowledgments

This work was supported by the Pharmaceutical Research Center and the Vice-Chancellor of Research, at Mashhad University of Medical Sciences.

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Trans-sodium crocetinate suppresses apoptotic and oxidative response following myoglobin-induced cytotoxicity in HEK-2 cells

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Cell culture studies

MB-induced cytotoxicity

Cell viability assay

Measurement of intracellular ROS generation

Western blot analysis

Statistical analysis

Results

Effect of MB on cell viability in HEK-2 cells

Effect of TSC on cell viability in HEK-2 cells

Effect of TSC on MB-induced cytotoxicity in HEK-2 Cells

Effect of TSC on MB-induced ROS generation in HEK-2 cells

Effect of MB and TSC on apoptotic factor in HEK-2 Cells

Effect of MB and TSC on autophagy proteins in HEK-2 cells

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1