Cardioprotective Effect of Spinosin against Doxorubicin Induced Inflammatory and Hypertrophy via Nrf2 Signaling

Mohammad Bani Ismail

College of Science, Department of Biological Sciences, King Faisal University, Al-Ahsa, 31982, Saudi Arabia.

ABSTRACT

Pathological cardiac remodeling can lead to heart failure and cardiac hypertrophy. Zizyphus jujuba var spinosa seeds contain SPN, a C-glycoside flavonoid used in traditional medicine. A growing body of evidence indicates they have pharmacological potential against a wide range of diseases. Spinosin (SPN), however, has not been examined in relation to doxorubicin (Dox) induced cardiac injuries. To find out if SPN can reverse Dox-induced cardiomyopathies, we conducted this study. Results showed that Dox treatment led to bigger cardiomyocytes, more inflammatory markers, and more cytokines. SPN treatment, however, reduced Dox-induced enlargement, attenuated hypertrophic markers, and attenuated the expression of inflammatory markers. Moreover, we saw increased levels of oxidative stress-related proteins NRF2, HO-1, and NQO-1. SPN protects against oxidative stress and inflammation-induced cardiomyopathies, according to our study.

Article Information

Received 16 December 2021

Revised 03 April 2022

Accepted 21 April 2022

Available online 01 August 2022

(early access)

Published 31 July 2023

Key words

Oxidative stress, SPN, Doxorubicin, Hypertrophy, Nrf2

DOI: https://dx.doi.org/10.17582/journal.pjz/20211216051231

* Corresponding author: mbaniismail@kfu.edu.sa

0030-9923/2023/0005-2181 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The most dangerous disease in the world is cancer, which is usually treated with doxorubicin (Dox) (Rivankar, 2014). In addition to causing cardiomyopathy, Dox also causes congestive heart failure (Huang et al., 2017; Mouli et al., 2015). In treating cancer patients with Dox, the biggest challenge is minimizing its cardiotoxic effects without compromising its antitumor capabilities. Dox’s cardiotoxicity remains a mystery. As underlying mechanisms, mitochondrial dysfunction, increased ROS production, defective iron handling, and contractile failure have been suggested (Alzahrani et al., 2021; Cheng et al., 2020). Dox also alters some signaling involved in regulating genes for vital processes, including metabolism and cell survival. In an effort to gain a better understanding of Dox-induced cardiac apoptosis, researchers have looked into the signaling pathways (Imam et al., 2018; Zhang et al., 2019). In this pathophysiology, oxidative stress plays a role in intracellular signaling and apoptosis. Through Nrf2 signaling via the Nfk pathway, this study tests whether spinosin (SPN) can protect against cardiac hypertrophy caused by Dox in cardiac cells.

Traditional medicine has used SPN, a C-glycoside flavonoid derived from the seeds of Zizyphus jujuba var spinosa, as a tranquilizer. SPN has been reported to have anxiolytic and hypnotic effects (Wang et al., 2012). SPN improves scopolamine- or amyloid beta oligomer-induced memory impairment in mice (Lee et al., 2016; Wang et al., 2000). SPN also penetrates the blood-brain barrier. However, it’s not clear whether SPN affects heart disease or improves it. SPN may be a promising therapeutic agent for cardiac disorders (Liu et al., 2015; Wang et al., 2000). Heart damage caused by Dox. With these factors in mind, the current study was designed to see if SPN can ameliorate Dox induced cardiomyopathies, and if so, what the mechanism is. SPN was found to significantly reduce Dox-induced cardiac hypertrophy, oxidative stress, and inflammation. SPN may be useful as a treatment for hypertension-induced heart damage because of its cardio protective properties.

Materials and Methods

Cell culture

In Dulbecco’s modified essential medium (DMEM, ThermoFisher Scientific, Waltham, MA, USA), we cultured H9c2 cardio myoblasts from the ATCC (VA, USA). H9c2 cells were treated with 100 mM (EC50) of SPN for 2 h before doxorubicin was added. We collected the cell lysate after 24 h.

MTT assay

H9c2 were cultured at 37°C with 10% FBS in DMEM. After 24 h, HUVECs were treated with Dox and/or SPN at the indicated concentrations. The MTT assay (Sigama, MO USA) was used to measure the proliferation of the cells.

Western blotting

We followed previously reported Western blotting procedures. Protein samples were separated on SDS-PAGE and then immunoblotted onto PVDF membranes. We developed the PBS-washed membranes and visualized the protein bands with a chemiluminescence substrate (Molqule-on Zwiterland). Samples were scanned with a LI-COR 3600-00-C-Digit Blot Scanner (AbuZahra et al., 2021). 

Actin staining

We stained the cells with actin staining for the analysis of hypertrophic effects on H9c2 cells, as we described in previous report (Rajendran et al., 2020).

Immunostaining

Following treatment, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature. In the next step, cells were permeabilized with 0.1% TritonX100 in PBS for 3–5 min, washed with PBS, and then incubated overnight with primary antibodies of Nrf2. Incubation with the primary antibodies was followed by three PBS washes and 1 h at RT in the dark with secondary antibodies (Lin et al., 2019).

ROS accumulation measurements

DCFH2-DA fluorescence dye was used to amount of intracellular ROS accumulation (Ismail et al., 2021). We seeded 1 X 107 cells/mL in a 6-well plate, then treated with SPN (for 2 h) followed by Dox treatment. Afterward, DCFH2-DA was supplementary to the culture medium for 30 min at 37°C. Using fluorescence microscopy, we examined the dichlorofluorescein (DCF) fluorescence intensity inside cells. By comparing the fluorescence intensity of treated cells and vehicle-treated cells, we measured ROS levels.

Tunel assay

As described earlier, we used the apoptosis detection TUNEL kit to analyze. In fluro microscopy at 454 nm, the nuclei of the TUNEL-positive cells were visible. We measured the images using microscopy (200X magnification) (Hu et al., 2019).

Statistical analysis

This study analyzed the data using an analysis of variance (one-way analysis of variance) and compared the controls using Tukey’s post-hoc test. The results were considered significant at p<0.05.

RESULTS

SPN protect cell death from Dox induced toxicity

MTT was used to test Dox and SPN for cytotoxicity. Figure 1A and B shows that treatment of HUVECs with SPN didn’t have any cytotoxic effects up to 100 µm/mL for 24 and 48 h. I used a SPN concentration of 100 µm/mL in the experiments. Figure 1C shows that Dox (0.5 and 1 µmol) significantly reduced cell viability up to 65%, but SPN dose-dependently protected the Dox-induced induction of cell death. It’s clear from these results that Dox is protective against Dox when exposed to cardiac toxicity.

 

Impact of SPN on Dox-induced hypertrophy 

By western blotting analysis, we first evaluated whether SPN could attenuate the expression of ANP and BNP. As shown in Figure 2A, treatment with Dox induces the expression of these proteins; however, treatment with SPN dose dependently reduces their expression. The expression of calcineurin, p-GATA-4, and GATA-4 (Fig. 2B) was assessed as well as the activation of hypertrophic signaling cascades related proteins. SPN also attenuated these signaling mediators, similar to the results above. Secondly, we determined whether SPN could reduce expansion of cardiomyocytes, in cardiac hypertrophy. Interesting, the results showed that Dox significantly enlarged cardiomyocytes, yet treatment with SPN significantly lowered the Dox-induced enlargement.

 

SPN and Dox-induced cardiac inflammation

 Furthermore, in view of the fact that inflammatory responses play a key role in the Dox-induced remodeling of the heart, we investigated whether SPN could dampen these inflammatory responses. We measured inflammatory cytokines like TNF-α, IL-6, and COX-2, as well as NF-кB expression (Fig. 3A). These inflammatory markers were significantly reduced by SPN.

Effects of SPN on Dox-induced cytoskeletal changes

The role of desmin and microtubule cytoskeletal alterations in cardiac hypertrophy and failure has been described in earlier experiments (Pai et al., 2018; Shibu et al., 2018). In the current study, we found that Dox increased cell size in H9c2 cardiomyoblasts within 24 h, as shown by the actin filament staining (Fig. 3B). F-actin staining showed SPN-treated cells reduced Dox-induced cellular hypertrophy after pretreatment. So, SPN inhibits Dox-induced cardiac hypertrophy in H9c2 cells.

 

 

Effect of SPN on Dox induced oxidative stress related proteins

Lastly, we examined the expression of oxidative stress-related proteins, such as Nrf2, HO-1, and NQO-1, in response to Dox induced cardiac remodeling (Fig. 4A). As can be seen, these oxidative stress markers were reduced after Dox treatment; however, SPN treatment upregulated their expression in a dose-dependent way. In Figure 4B visualized the nuclear localization of Nrf2 in H9c2 with SPN using fluorescence microscopy. According to immunofluorescence images, as seen by the high Nrf2 staining in SPN-treated cells, SPN increased Nrf2 nuclear aggregation. ROS level significantly elevated in Dox treated cells in fig 4C, whereas SPN with Dox cells dramatically reduce this elevated level of ROS.

SPN inhibit apoptosis in Dox induced H9c2 cells

We looked at Western blots to see if SPN activates Bcl-2 and Bcl-xL and caspase-3 in H9c2s in Dox treated cells. In Dox with SPN therapy, Bcl-2 and Bcl-xL levels increased and caspase-3 levels decreased (Fig. 5A). SPN activated Bcl-2 and Bcl-xL and suppressed apoptosis. To investigate how SPN prevents Dox-induced cell death, we treated endothelial cells with SPN and/or Dox for 24 h with DNA fragmentation measured with TUNEL (Fig. 5B). The fluorescence microscopy images showed an increase in the number of TUNEL-positive cells after Dox treatment alone, whereas SPN treatment reduced TUNEL-positive cells significantly. SPN treatment clearly prevents Dox-induced apoptosis in H9c2 cells.

 

Discussion

This study examined the cardio-protective role of SPN against Dox-induced cardiac injury. Researchers are trying to understand the pathophysiology behind cardiac injuries. There is a lot of consensus that a better understanding of this complex mechanism might lead to new avenues of prevention and/or treatment for hypertension-induced cardiomyopathies (Barik et al., 2021; Lin et al., 2021a, b). It is widely accepted that contractile dysfunction in cardiomyocytes is the result of a combination of factors including hypertrophy in the heart, oxidative stress, and inflammatory responses. In other words, attenuating these underlying responses might help preserve the contractile function of the cardiomyocytes, which might cardiomyocytes. This even prevents cardiac dysfunction. Hypertrophy of cardiomyocytes often causes heart failure. Sympathetic hyperactivity and Dox-induced hypertension can result from an imbalance between pro- and anti-inflammatory cytokines (Pecoraro et al., 2016; Quagliariello et al., 2021; Singla et al., 2019). Western blot analysis of myocardial cells in the Dox group revealed elevated COX-2, TNF-α, and IL-6 cytokines. A significant increase in myocardial inflammation accompanied the elevated inflammatory cytokines. Many anti-inflammatory drugs have been reported to protect against Dox-induced myocardial damage, suggesting inflammation plays a big part. SPN has been found to have significant anti-inflammatory potential, and it restored the normal inflammatory cytokine blance (Lee et al., 2016; Xu et al., 2019). SPN blocks doxorubicin-induced cytokine depletion. Several pathological events cause the myocardium to hypertrophy, including myocardial infarction, hypertension, and adrenal over-activity. Cell size increases, protein synthesis increases, and ANP, BNP, calcinurin and phosphorilation of GATA4 fetal cardiac genes get stimulated (Lin et al., 2019). Current findings confirm that Dox induces a cardiac hypertrophic response, correlated with increases in ANP and BNP, markers of hypertrophy. In H9c2 cells treated with SPN, there was a significant decrease in levels of the above-mentioned proteins, which prevented hypertrophy induced by Dox. Therefore, SPN has a protective effect on cardiac hypertrophy.

Nrf2 is a transcription factor that regulates a bunch of cyto protective genes in the heart. Through attenuating oxidative stress, activating Nrf2 has been shown to protect against pathological cardiac remodeling. Further studies showed that SPN increased Nrf2 levels, indicating its ability to amplify Nrf2 pathway, thereby suppressing oxidative stress, which is critical for HF development. So, it’s possible to envision that SPN might play a role in restoring the redox balance, which then orchestrates events that help with heart remodeling. Inflammatory responses play an important role in Dox-induced cardiac remodeling. Interestingly, the SPN reduces the expression of these inflammatory cytokines COX-2, IL-6, TNF-α and NF-кB by a various dose- reliant on mechanism. Bcl-2 and Bcl-xL inhibit cytochrome c release and protect cardiac cells from oxidative stress and apoptosis (Chen et al., 2017). Rather, caspase-3 executes the apoptotic program and cleaves PARP when the cell dies (Chen et al., 2017). In our study, we found that SPN treatment induced upregulation of Bcl2 and Bcl-xL, and suppressed Dox-induced downregulation of Bcl2. However, SPN suppressed caspase activation.

Combined, our study shows that SPN protects against Dox-induced cardiomyopathies. In vitro and animal testing still needs to be done to define and illuminate the rationale behind its use in the clinic.

Acknowledgments

I acknowledge with thanks the Deanship of Scientific Research at King Faisal University, Saudi Arabia for financial support under grant number 170058.

Data availability statement

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

Statement of conflict of interest

The authors have declared no conflict of interest.

References

AbuZahra, H.M., Rajendran, P., and Ismail, M.B., 2021. Zerumbone exhibit protective effect against zearalenone in-duced toxicity via ameliorating inflammation and oxidative stress induced apoptosis. Antioxidants 10: 1593. https://doi.org/10.3390/antiox10101593

Alzahrani, A.M., Rajendran, P., Veeraraghavan, V.P., and Hanieh, H., 2021. Cardiac protective effect of kirenol against doxorubicin-induced cardiac hypertrophy in H9c2 cells through Nrf2 signaling via PI3K/AKT pathways. Int. J. mol. Sci., 22: 3269. https://doi.org/10.3390/ijms22063269

Barik, P., Shibu, M.A., Hsieh, D.J.Y., Day, C.H., Chen, R.J., Kuo, W.W., Chang, Y.M., Padma, V.V., Ho, T.J., and Huang, C.Y., 2021. Cardioprotective effects of transplanted adipose-derived stem cells under Ang II stress with Danggui administration augments cardiac function through upregulation of insulin-like growth factor 1 receptor in late-stage hypertension rats. Environ. Toxicol., 36: 1466-1475. https://doi.org/10.1002/tox.23145

Chen, M., Cai, F., Zha, D., Wang, X., Zhang, W., He, Y., Huang, Q., Zhuang, H., and Hua, Z.C., 2017. Astragalin-induced cell death is caspase-dependent and enhances the susceptibility of lung cancer cells to tumor necrosis factor by inhibiting the NF-κB pathway. Oncotarget, 8: 26941. https://doi.org/10.18632/oncotarget.15264

Cheng, D., Chen, L., Tu, W., Wang, H., Wang, Q., Meng, L., Li, Z., and Yu, Q., 2020. Protective effects of valsartan administration on doxorubicin-induced myocardial injury in rats and the role of oxidative stress and NOX2/NOX4 signaling. Mol. Med. Rep., 22: 4151-4162. https://doi.org/10.3892/mmr.2020.11521

Hu, J., Wu, Q., Wang, Z., Hong, J., Chen, R., Li, B., Hu, Z., Hu, X., and Zhang, M., 2019. Inhibition of CACNA1H attenuates doxorubicin-induced acute cardiotoxicity by affecting endoplasmic reticulum stress. Biomed. Pharmacother., 120: 109475. https://doi.org/10.1016/j.biopha.2019.109475

Huang, L., Zhang, K., Guo, Y., Huang, F., Yang, K., Chen, L., Huang, K., Zhang, F., Long, Q., and Yang, Q., 2017. Honokiol protects against doxorubicin cardiotoxicity via improving mitochondrial function in mouse hearts. Sci. Rep., 7: 1-12. https://doi.org/10.1038/s41598-017-12095-y

Imam, F., Al-Harbi, N.O., Al-Harbi, M.M., Ansari, M.A., Al-Asmari, A.F., Ansari, M.N., Al-Anazi, W.A., Bahashwan, S., Almutairi, M.M., and Alshammari, M., 2018. Apremilast prevent doxorubicin-induced apoptosis and inflammation in heart through inhibition of oxidative stress mediated activation of NF-κB signaling pathways. Pharmacol. Rep., 70: 993-1000. https://doi.org/10.1016/j.pharep.2018.03.009

Ismail, M.B., Rajendran, P., AbuZahra, H.M., and Veeraraghavan, V.P., 2021. Mangiferin inhibits apoptosis in doxorubicin-induced vascular endothelial cells via the Nrf2 signaling pathway. Int. J. mol. Sci., 22: 4259. https://doi.org/10.3390/ijms22084259

Lee, Y., Jeon, S.J., Lee, H.E., Jung, I.H., Jo, Y.W., Lee, S., Cheong, J.H., Jang, D.S., and Ryu, J.H., 2016. SPN, a C-glycoside flavonoid, enhances cognitive performance and adult hippocampal neurogenesis in mice. Pharmacol. Biochem. Behav., 145: 9-16. https://doi.org/10.1016/j.pbb.2016.03.007

Lin, C.Y., Shibu, M.A., Wen, R., Day, C.H., Chen, R.J., Kuo, C.H., Ho, T.J., Viswanadha, V.P., Kuo, W.W., and Huang, C.Y., 2021. Leu27IGF-II-induced hypertrophy in H9c2 cardiomyoblasts is ameliorated by saffron by regulation of calcineurin/NFAT and CaMKIIδ signaling. Environ. Toxicol., 36: 2475-2483. https://doi.org/10.1002/tox.23360

Lin, K.H., Kumar, V.B., Shanmugam, T., Shibu, M.A., Chen, R.J., Kuo, C.H., Ho, T.J., Padma, V.V., Yeh, Y.L., and Huang, C.Y., 2021. miR-145-5p targets paxillin to attenuate angiotensin II-induced pathological cardiac hypertrophy via downregulation of Rac 1, pJNK, pc-Jun, NFATc3, ANP and by Sirt-1 upregulation. Mol. Cell. Biochem., pp. 1-8. https://doi.org/10.1007/s11010-021-04100-w

Lin, K.H., Shibu, M.A., Peramaiyan, R., Chen, Y.F., Shen, C.Y., Hsieh, Y.L., Chen, R.J., Viswanadha, V.P., Kuo, W.W., and Huang, C.Y., 2019. Bioactive flavone fisetin attenuates hypertension associated cardiac hypertrophy in H9c2 cells and in spontaneously hypertension rats. J. Funct. Fds., 52: 212-218. https://doi.org/10.1016/j.jff.2018.10.038

Liu, J., Zhai, W.M., Yang, Y.X., Shi, J.L., Liu, Q.T., Liu, G.L., Fang, N., Li, J., and Guo, J.Y., 2015. GABA and 5-HT systems are implicated in the anxiolytic-like effect of SPN in mice. Pharmacol. Biochem. Behav. 128: 41-49. https://doi.org/10.1016/j.pbb.2014.11.003

Mouli, S., Nanayakkara, G., AlAlasmari, A., Eldoumani, H., Fu, X., Berlin, A., Lohani, M., Nie, B., Arnold, R.D., and Kavazis, A., 2015. The role of frataxin in doxorubicin-mediated cardiac hypertrophy. Am. J. Physiol. Heart Circ. Physiol., 309: H844-H859. https://doi.org/10.1152/ajpheart.00182.2015

Pai, P., Shibu, M.A., Chang, R.L., Yang, J.J., Su, C.C., Lai, C.H., Liao, H.E., Viswanadha, V.P., Kuo, W.W., and Huang, C.Y., 2018. ERβ targets ZAK and attenuates cellular hypertrophy via SUMO-1 modification in H9c2 cells. J. Cell. Biochem., 119: 7855-7864. https://doi.org/10.1002/jcb.27199

Pecoraro, M., Del Pizzo, M., Marzocco, S., Sorrentino, R., Ciccarelli, M., Iaccarino, G., Pinto, A., and Popolo, A., 2016. Inflammatory mediators in a short-time mouse model of doxorubicin-induced cardiotoxicity. Toxicol. appl. Pharmacol., 293: 44-52. https://doi.org/10.1016/j.taap.2016.01.006

Quagliariello, V., De Laurentiis, M., Rea, D., Barbieri, A., Monti, M.G., Carbone, A., Paccone, A., Altucci, L., Conte, M., and Canale, M.L., 2021. The SGLT-2 inhibitor empagliflozin improves myocardial strain, reduces cardiac fibrosis and pro-inflammatory cytokines in non-diabetic mice treated with doxorubicin. Cardiovasc. Diabetol., 20, 1-20. https://doi.org/10.1186/s12933-021-01346-y

Rajendran, P., Ben Ammar, R., Al-Saeedi, F. J., Elsayed Mohamed, M., Islam, M., and Al-Ramadan, S.Y., 2020. Thidiazuron decreases epithelial-mesenchymal transition activity through the NF-kB and PI3K/AKT signalling pathways in breast cancer. J. cell. mol. Med., 24: 14525-14538. https://doi.org/10.1111/jcmm.16079

Rivankar, S., 2014. An overview of doxorubicin formulations in cancer therapy. J. Cancer Res. Ther., 10: 853. https://doi.org/10.4103/0973-1482.139267

Shibu, M.A., Kuo, C.H., Chen, B.C., Ju, D.T., Chen, R.J., Lai, C.H., Huang, P.J., Viswanadha, V.P., Kuo, W.W., and Huang, C.Y., 2018. Oolong tea prevents cardiomyocyte loss against hypoxia by attenuating p-JNK mediated hypertrophy and enhancing P-IGF1R, p-akt, and p-Badser136 activity and by fortifying NRF2 antioxidation system. Environ. Toxicol., 33: 220-233. https://doi.org/10.1002/tox.22510

Singla, D.K., Johnson, T.A., and Tavakoli, D.Z., 2019. Exosome treatment enhances anti-inflammatory M2 macrophages and reduces inflammation-induced pyroptosis in doxorubicin-induced cardiomyopathy. Cells, 8: 1224. https://doi.org/10.3390/cells8101224

Wang, L.E., Zhang, X.Q., Yin, Y.Q., and Zhang, Y.H., 2012. Augmentative effect of SPN on pentobarbital-induced loss of righting reflex in mice associated with presynaptic 5-HT1A receptor. J. Pharm. Pharmacol., 64: 277-282. https://doi.org/10.1111/j.2042-7158.2011.01400.x

Wang, S., Scott, B.W., and Wojtowicz, J.M., 2000. Heterogenous properties of dentate granule neurons in the adult rat. J. Neurobiol., 42: 248-257. https://doi.org/10.1002/(SICI)1097-4695(20000205)42:2<248::AID-NEU8>3.0.CO;2-J

Xu, F., He, B., Xiao, F., Yan, T., Bi, K., Jia, Y., and Wang, Z., 2019. Neuroprotective effects of SPN on recovery of learning and memory in a mouse model of Alzheimer’s disease. Biomol. Ther., 27: 71. https://doi.org/10.4062/biomolther.2018.051

Zhang, X., Zhu, J.X., Ma, Z.G., Wu, H.M., Xu, S.C., Song, P., Kong, C.Y., Yuan, Y.P., Deng, W., and Tang, Q.Z., 2019. Rosmarinic acid alleviates cardiomyocyte apoptosis via cardiac fibroblast in doxorubicin-induced cardiotoxicity. Int. J. biol. Sci., 15: 556. https://doi.org/10.7150/ijbs.29907