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. 2024;27(6):768-774.
doi: 10.22038/IJBMS.2024.75306.16322.

Trans-sodium crocetinate suppresses apoptotic and oxidative response following myoglobin-induced cytotoxicity in HEK-293 cells

Tahereh Aminifard  1 , Soghra Mehri  1   2 , Mahboobeh Ghasemzadeh Rahbardar  2 , Fatemeh Rajabian  1 , Abolfazl Khajavi Rad  3 , Hossein Hosseinzadeh  1   2
Affiliations

Trans-sodium crocetinate suppresses apoptotic and oxidative response following myoglobin-induced cytotoxicity in HEK-293 cells

Tahereh Aminifard et al. Iran J Basic Med Sci. 2024.

Abstract

Objectives: Rhabdomyolysis (RM) is a serious fatal syndrome. The RM leads to acute kidney injury (AKI) as a fatal complication. The belief is that RM-induced AKI is triggered by myoglobin (MB). MB activates oxidative and apoptotic pathways. Trans-sodium crocetinate (TSC) is obtained from saffron. It has anti-oxidant and renoprotective effects. This research was designed to assess the mechanisms of MB-induced cytotoxicity in HEK-293 cells (human embryonic kidney cells) as well as the possible effects of TSC against MB-induced cytotoxicity.

Materials and methods: HEK-293 cells were exposed to diverse concentrations of TSC (2.5, 5, 10, 20, 40, 80, and 100 µM) for 24 hr. Then, MB (9 mg/ml) was added to the cells. After 24 hr, cell viability was measured through MTT, and the values of ROS generation were calculated using DCFH-DA assay. Also, autophagy and apoptosis markers in cells were assessed by western blot analysis.

Results: MB decreased viability and increased ROS levels in HEK-293 cells. However, pretreatment of HEK-293 cells with TSC for 24 hr reduced the cytotoxicity and ROS production caused by MB. Furthermore, MB enhanced both the apoptosis (cleaved caspase-3 and Bax/Bcl-2 ratio) and autophagy markers (LC3II/I ratio and Beclin-1) in HEK-293 cells. On the other hand, TSC pretreatment condensed the levels of autophagy and apoptosis criteria in response to MB cytotoxicity.

Conclusion: TSC has a positive effect in preventing MB-induced cytotoxicity in HEK-293 cells by increasing anti-oxidant activity and regulation of apoptotic and autophagy signaling pathways.

Keywords: Acute Kidney injuries; Apoptosis; Autophagy; Myoglobin; Rabdomyolysis; Trans-sodium crocetinate.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Effect of MB on HEK-293 cell viability evaluated by MTT assay. Data are presented as mean±SD of four separate experiments. Data were analyzed by one-way ANOVA and the Tukey-Kramer post-test. ***P<0.001 and*P<0.05 vs control-treated cells
Figure 2
Figure 2
Effect of TSC on HEK-293 cell viability evaluated by MTT assay. Data are presented as mean±SD of four separate experiments. Data were analyzed by one-way ANOVA
Figure 3
Figure 3
Effect of TSC on MB-induced toxicity on HEK-293 cells evaluated by MTT assay. Data are presented as mean±SD of four separate experiments. Data were analyzed by one-way ANOVA and the Tukey-Kramer post-test. ***P<0.001 vs control group, ###P<0.001 vs MB (9 mg/ml) treated cells
Figure 4
Figure 4
Effect of TSC on MB-induced ROS generation in HEK-293 cells. Data are presented as mean±SD of three separate experiments. Data were analyzed using one-way ANOVA and the Tukey-Kramer post-test. ###P<0.001 vs control group and ***P<0.001 and **P<0.01 vs MB (9 mg/ml) group
Figure 5
Figure 5
Effect of MB and TSC on Bax/Bcl-2 ratio in HEK-293 cells
Figure 6
Figure 6
Effect of MB and TSC on cleaved caspase-3 expression in HEK-293 cells
Figure 7
Figure 7
Effect of MB and TSC on Beclin-1 expression in HEK-293 cells
Figure 8
Figure 8
Effect of MB and TSC on LC3 II and LC3 I expression in HEK-293 cells
Figure 9
Figure 9
Protective effect of TSC on MB-induced cytotoxicity in HEK-293 cells via oxidative stress, autophagy, and apoptosis signaling pathways
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