Fisetin inhibits patulin-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12

doi:10.21203/rs.3.rs-4839276/v1

Download PDF

Research Article

Fisetin inhibits patulin-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12

https://doi.org/10.21203/rs.3.rs-4839276/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Objectives of the Study

Fisetin (FIS) has a good protective effect on the heart. However, fisetin in regulating the role of the myocardial injury induced by patulin (PAT) is not clear. The aim of this study is to investigate the possible mechanism of fisetin in attenuating patulin induced myocardial injury.

Materials and Methods

Cardiomyocytes were treated with 25μM PAT to set the control group, FIS only group, PAT only group and PAT-FIS addition group. LDH activity, SOD content, and MDA content were evaluated using kits. ROS levels were determined by measuring the intensity of fluorescence. Mitochondrial membrane potential was detected by JC-1 dye staining. The protein expressions of Grp78, Chop and Caspase-12 were detected by Western blot.

Results

In PAT-FIS group, LDH release and MDA content decreased, but SOD content increased. Compared with the control group, the level of ROS in PAT group increased more than 10 times. The level of ROS in the PAT-FIS group was still higher than that in the control group, but it was significantly lower than that in the PAT group. The proportion of red fluorescence in the mitochondrial membrane potential of cardiomyocytes increased from 75% to 85% in the PAT-FIS group. PAT up-regulated the expression of Chop, Grp78 and Caspase-12 proteins, while the overexpression of Chop, Grp78 and Caspase-12 proteins was inhibited after pretreatment with FIS and PAT .

Conclusion

Our findings suggest that FIS inhibits PAT-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12 signaling.

patulin

fisetin

myocardial damage

Grp78

Chop

Caspase-12

Fisetin(C₁₅H₁₀O₆, FIS) is a naturally occurring flavonoid, found in a variety of fruits (mangoes, apples, strawberries, kiwifruit and grapes), vegetables (cucumbers, tomatoes and Onions), nuts and wine [1]. Studies have shown that FIS has many beneficial properties, including anti-cancer, anti-clotting, anti-inflammatory and antioxidant effects[2]. We have previously shown that FIS can inhibit PAT-induced cardiomyocyte apoptosis by inhibiting Caspase3/8/9, P53, and Bcl-2/Bax targets [3]. In addition, FIS has been shown to inhibit ROS/ER stress-mediated inflammatory responses, helping to improve cardiac damage induced by metabolic stimuli in vivo and in vitro [4]. FIS alleviates hypertension-related cardiac hypertrophy in H9c2 cells and spontaneously hypertensive rats [5]. FIS also plays a cardioprotective role in ameliorating oxidative stress, inflammation, and apoptosis in diabetic cardiomyopathy induced by HFD or streptozotocin (STZ) [6] [7].

Patulin (PAT) is a mycotoxin produced primarily by Penicillium expansum. Patulin was first found in mouldy apples and apple juice, and mainly contaminated fruits and their products, especially apples, hawthorns, pears, tomatoes, apple juice and hawthorn slices [8]. In addition, patulin contamination also exists in some Chinese medicinal materials (codonopsis, astragalus, etc.) and feed [9]. Toxicological studies of PAT have shown that ingestion of PAT affects a variety of organs such as the brain, liver and kidney in experimental animals [10] [11] [12]. Long-term exposure also has neurotoxic, immunotoxic, genotoxic and teratogenic effects [13]. After entering cardiomyocytes, PAT induces oxidative stress and inflammatory response of cardiomyocytes, resulting in apoptosis of cardiomyocytes, myocardial toxicity and cardiac injury [14].

As mentioned above, PAT can induce severe toxicity, and FIS is a promising natural extract for heart protection. However, as far as we know, the inhibitory mechanism of FIS on PAT is not very clear. Therefore, in this study, the contents of LDH, SOD, MDA, ROS and mitochondrial membrane potential in H9c2 cardiomyocytes were measured. Finally, the possible detoxification mechanism of FIS was explored by detecting the expressions of Grp78, Chop and Caspase-12 proteins in H9c2 cardiomyocytes.

2.1 Materials and Reagents

Patulin (CAS: 149-29-1, purity ≥ 99%) was purchased from Sigma-Aldrich (Shanghai, China). Fisetin (CAS: 528-48-3, purity ≥ 98%) was purchased from AbMole. BCA protein detection kit, IP cell lysis buffer, phenylmethyl sulfonyl fluoride (PMSF) were obtained from Beyotime (Shanghai, China). Polyvinylidene fluoride (PVDF) membranes with an average pore size of 0.45µm were supplied by Millipore (St. Louis, USA). LDH test kit, MDA test kit, acrylamide, double acrylamide, triple, glycine and ammonium persulfate (AP) solution were obtained from Solarbio (Beijing, China). SOD detection kit and ROS detection kit were obtained from Nanjing Jiancheng Biological Company. The JC-1 mitochondrial membrane Potential (MMP) test kit was purchased from US EVERBRIGHT INC. SDS-PAGE Loading buffer (5x) was purchased from CWBIO (Beijing, China). All of the above chemicals are standard analytical grade or higher.

2.2 The contents of LDH, SOD and MDA in cells were detected

H9c2 cardiomyocytes were purchased from the Laboratory of the Chinese Academy of Sciences in Shanghai. Cells were routinely cultured with 10%FBS and 1% penicillin-streptomycin in an incubator at 37°C and 5%CO₂. According to our previous study [3], cardiomyocytes were treated with 25µM PAT in a control group, FIS only group, PAT only group, and PAT-FIS addition group. After cell culture and treatment, refer to the instructions of LDH test kit, SOD test kit and MDA test kit respectively for operation.

2.3 H9c2 cardiomyocytes ROS content determination

The final concentration of DCFH-DA was 10 µM after 1000 times dilution in serum-free medium. Dilute DCFH-DA was added into the cells and incubated at 37℃ for 20 min. After 20 min, DCFH-DA was absorbed and the cells were washed without serum culture solution, and this was repeated three times. Then, the cells were collected and transferred into the flow tube. The fluorescence intensity before and after stimulation was detected in real time by cytometry at excitation wavelength of 488 nm and emission wavelength of 525 nm.

2.4 Mitochondrial membrane potential (MMP) determination

Take an appropriate amount of JC-1 (200×) and dilute JC-1 by adding 8 mL of ultra-pure water every 50 µL JC-1 (200×). Fully dissolve and mix JC-1 by violent shock. Then add 2 mL JC-1 dyeing buffer (5 ×), and mix it into JC-1 dyeing working solution. The treated cells were removed from the incubator, digested into a centrifuge tube and centrifuged at 400 g for 5 min. After the supernatant was removed, the culture solution was re-suspended, and JC-1 working solution was added according to the ratio of 1:1 between the culture solution and JC-1 working solution, and incubated at 37℃ for 20 min. After incubation at 37℃, centrifuge 600 g at 4℃ for 3 ~ 4 min and discard the supernatant. The cells were washed twice with JC-1 buffer, then re-suspended with JC-1 buffer and detected by flow cytometry. The data is post-processed by Flow Jo software.

2.5 Western blot analysis

Protein was extracted using RIPA lysis and Extraction buffer (Solarbio). The protein content was determined using the BCA protein assay kit (Beyotime). The extracted proteins (40µg) were placed in a 10–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) system and then transferred to PVDF membranes (Millipore Company, USA). Next, close the membrane with 5% skim milk for 1 hour. They were incubated with primary antibodies at 4℃ overnight. Wash the PVDF membrane. After that, the cells were incubated with the second antibody for 1.5 hours. After the second antibody incubation, PVDF membrane was removed and washed for 3 times. An appropriate amount of luminescent liquid was added to the PVDF membrane, and then placed in the gel imaging system, exposed, and photographed. Image J software was used for gray scale analysis. The ratio of target band to internal reference band was taken as the result, the ratio of samples in each treatment group to blank control indicated the protein level, and the value of control group was set to 1.

2.6 Statistical analysis

The result is expressed as "mean ± standard deviation". The Graphpad prism 9.5 software was used for significance analysis, and P < 0.05 indicated a significant difference, p < 0.01, denoted by "**".

3.1 FIS inhibits oxidative stress induced by PAT

Compared with Control, LDH release in PAT group was increased by 2.5 times (p < 0.05). Compared with PAT group, LDH release was significantly reduced in FIS+PAT group (p < 0.001) (Fig.1A). SOD activity in FIS group (20Μm FIS and 30μM FIS) had no significant change, but SOD activity in PAT group was significantly decreased compared with blank group (p < 0.001), and SOD activity in PAT group could only reach 1/2 of that in blank group (Fig.1B). Compared with PAT group, SOD content in FIS treatment group (25μM PAT+ 20μM FIS and 25μM PAT+ 30μM FIS) was significantly increased, with statistical significance. There was no significant difference in MDA content between control group and FIS group. MDA content in 25μM PAT group was significantly increased, reaching 6 times of that in control group. After FIS treatment, MDA content decreased significantly compared with 25μM PAT group, that is, MDA content in PAT +FIS group decreased by 4 times as much as that in control group (Fig.1C). These results indicated that cells were more sensitive to PAT stimulation, and oxidative damage occurred in cells, which seriously affected lipid oxidation and increased MDA content. However, FIS treatment decreased MDA content.

3.2 FIS pretreatment reduced PAT-induced ROS levels

There was no significant difference in ROS intensity between 20μM FIS and 30μM FIS groups compared with control group (Fig.2). Compared with the control group, the fluorescence intensity and ROS level in PAT group were increased by more than 10 times. After FIS pretreatment, although the ROS level was still higher than that of the control group, the ROS level was significantly lower than that of the PAT group. Therefore, we can boldly speculate that PAT can cause cells to produce excess and harmful ROS, and then induce cell oxidative damage, leading to apoptosis and necrosis; However, FIS treatment can effectively inhibit the toxic effect of PAT and enhance the ability of cells to resist foreign stimuli.

3.3 The effect of PAT on mitochondrial membrane potential of cardiomyocytes decreased after FIS treatment

The results were analyzed by a 2-dimensional scatter plot, which was divided into four quadrants by a cross gate. The upper right quadrant (UR) shows that JC-1 exists in the cell in polymer form and fluoresces red. The lower right quadrant indicates that JC-1 is present in cells as a monomer and emits green fluorescence. When the mitochondrial membrane potential is reduced, JC-1 switches from red to green fluorescence, a hallmark signal of early apoptosis. After PAT treatment, the mitochondrial membrane permeability transport pore (MPTP) of H9c2 cardiomyocytes was opened, and the internal and external charge of mitochondria was disordered, which was manifested as a significant increase in green fluorescence in the PAT group compared with the control group (Fig.3). However, FIS pretreatment reduced the effect of PAT on mitochondrial membrane potential, and the proportion of red fluorescence in the mitochondrial membrane potential increased from 75% in PAT group to 85% in FIS group.

3.4 FIS inhibits PAT-induced cardiomyocyte apoptosis by regulating Grp78/Chop/Caspase-12

After ER stress is activated, Grp78 is released as a key ER stress sensor [15]. Severe ER stress can promote the expression of pro-apoptotic proteins, such as Chop [16]. Finally, cells undergo apoptotic cell death under stimulating conditions [15]. Caspase-12, Grp78 and Chop, as important markers of endoplasmic reticulum stress-mediated apoptosis, play a key role in the apoptosis process [17]. The expressions of apoptosis-related proteins Caspase-12, Chop and Grp78 were detected by Western blotting. The ratio of apoptosis-related proteins to β-actin was quantified by ImageJ software. As shown in Figure 4, PAT up-regulated the expression levels of Chop, Grp78, and Caspase-12 proteins; while adding PAT after FIS pretreatment inhibited the overexpression of Chop, Grp78, and Caspase-12 proteins compared with adding PAT only (p < 0.001).

Apoptosis is an active process of cell death, involving two pathways: mitochondrial pathway and membrane receptor pathway [18]. The endoplasmic reticulum plays an important role in a variety of cellular processes required for cell survival and normal cell function, including intracellular calcium homeostasis, protein secretion and folding of secreted proteins, lipid biosynthesis, and it is involved in the intrinsic pathway of apoptosis [19]. At rest, Grp78 binds to an electrorheological membrane protein. However, under stimulated conditions, Grp78 will be released from endoplasmic reticulum proteins, resulting in homologous oligomerization of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol dependent enzyme 1 (IRE1), and a large number of unfolded proteins accumulate in the endoplasmic reticulum [20]. Activation of the transcription factor C/EBP homologous protein (CHOP) triggers the endoplasmic reticulum to cause pro-apoptotic signaling, thereby promoting the activation of pro-apoptotic proteins and leading to apoptosis [21].

Endoplasmic reticulum stress may also lead to oxidative stress, resulting in toxic accumulation of ROS within cells [22]. ROS play a key role in processes such as cell signaling, stress response and cell death [23] [24]. There is a close association between mitochondrial membrane potential and ROS [25]. When cells are under oxidative stress, a large number of reactive oxygen species (ROS) are produced in mitochondria, which will lead to a decrease in mitochondrial membrane potential. At the same time, the decrease of mitochondrial membrane potential further increases ROS production. In our study, PAT caused oxidative stress in cardiomyocytes with increased ROS. And FIS can significantly inhibit oxidative stress caused by PAT, and inhibiting ROS generation, make PAT has less of an effect on myocardial cell mitochondrial membrane potential. PAT treated H9c2 cardiomyocytes showed significantly increased protein expressions of Grp78 and Chop. FIS prevented the pro-apoptotic process by down-regulating Grp78 and Chop, and partially protected H9c2 cardiomyocytes.

WB results showed that FIS could significantly down-regulate the overexpression of Caspase-12 protein induced by PAT and block the transmission of apoptosis signal. Caspase-12 is located in ER and is considered to be a specific apoptotic mediator in the ERS apoptosis pathway [26]. In the ERS state, the increase of intracellular calcium ions leads to cytoplasmic activation of calpsin, and the precursor of Caspase-12 located on the ER membrane is cut for activation and release into the cytoplasm [27]. After Caspase-12 is activated, Caspase-9 is activated, and Caspase-9 activates downstream Caspase-3, initiating the classical apoptotic pathway and ultimately leading to apoptosis [28].

In conclusion, there are two main findings from the current study. Firstly, we demonstrated that FIS treatment could alter PAT-induced oxidative stress, ROS content and MMP in cardiomyocytes. Secondly, we detected the protein expression of Caspase12, Chop, and Grp78 to confirm the cardioprotective effect of FIS treatment.

Compliance with Ethical Standards

Funding: This work was supported by the National Natural Science Foundation of China (31760495); the Nature Fund of Gansu Province(18JR3RA136); Qingdao Institute of Technology Field of Scientific Research Projects.

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Ackonwledgements

This work was supported by the National Natural Science Foundation of China (31760495); the Nature Fund of Gansu Province(18JR3RA136); Qingdao Institute of Technology Field of Scientific Research Projects.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Baigang Zhang: Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Funding support. Dongmei Xu: Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-review & editing. Chenghui Huang: Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-review & editing. Lu Jiao: Formal analysis, Investigation, Methodology, Software， Investigation, Methodology, Validation, Writing-original draft, Writing - review & editing. Yang Li: Methodology, Visualization, Formal analysis, Software, Investigation, Project administration. Binggang Fu: Formal analysis, investigation, visualization, software, verification. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

G. Grynkiewicz and O. M. Demchuk, "New Perspectives for Fisetin," (in English), Frontiers in Chemistry, Review vol. 7, 2019-October-30 2019.
L. Long et al., "Protective effects of fisetin against myocardial ischemia/reperfusion injury," (in eng), Experimental and Therapeutic Medicine, vol. 19, no. 5, pp. 3177–3188, 2020.
D. Xu, B. Zhang, C. Huang, J. Lu, Y. Li, and B. Fu, "Effect and mechanism of Fisetin on myocardial damage induced by Patulin," (in eng), Molecular Biology Reports, vol. 50, no. 8, pp. 6579-6589, 2023.
C.-X. Ge et al., "Endoplasmic reticulum stress-induced iRhom2 up-regulation promotes macrophage-regulated cardiac inflammation and lipid deposition in high fat diet (HFD)-challenged mice: Intervention of fisetin and metformin," Free Radical Biology and Medicine, vol. 141, pp. 67-83, 2019/09/01/ 2019.
K.-H. Lin et al., "Bioactive flavone fisetin attenuates hypertension associated cardiac hypertrophy in H9c2 cells and in spontaneously hypertension rats," Journal of Functional Foods, vol. 52, pp. 212-218, 2019/01/01/ 2019.
L.-F. Hu et al., "Oral flavonoid fisetin treatment protects against prolonged high-fat-diet-induced cardiac dysfunction by regulation of multicombined signaling," The Journal of Nutritional Biochemistry, vol. 77, p. 108253, 2020/03/01/ 2020.
O. Y. Althunibat, A. M. Al Hroob, M. H. Abukhalil, M. O. Germoush, M. Bin-Jumah, and A. M. Mahmoud, "Fisetin ameliorates oxidative stress, inflammation and apoptosis in diabetic cardiomyopathy," Life Sciences, vol. 221, pp. 83-92, 2019/03/15/ 2019.
M. Xing, Y. Chen, B. Li, and S. Tian, "Characterization of a short-chain dehydrogenase/reductase and its function in patulin biodegradation in apple juice," Food Chemistry, vol. 348, p. 129046, 2021/06/30/ 2021.
M. A. Al-Hazmi, "Patulin in apple juice and its risk assessments on albino mice," Toxicology and Industrial Health, vol. 30, no. 6, pp. 534-545, 2014.
C. Wei et al., "Progress in the distribution, toxicity, control, and detoxification of patulin: A review," Toxicon, vol. 184, pp. 83-93, 2020/09/01/ 2020.
B. Zhang, C. Huang, Q. Lu, H. Liang, J. Li, and D. Xu, "Involvement of caspase in patulin-induced hepatotoxicity in vitro and in vivo," Toxicon, vol. 206, pp. 64-73, 2022/01/30/ 2022.
O. Puel, P. Galtier, and I. P. Oswald, "Biosynthesis and Toxicological Effects of Patulin," Toxins, vol. 2, no. 4, pp. 613-631. doi: 10.3390/toxins2040613
W.-L. Lei et al., "Toxic effects of patulin on mouse oocytes and its possible mechanisms," Toxicology, vol. 464, p. 153013, 2021/12/01/ 2021.
B. Zhang et al., "Cardiotoxicity of patulin was found in H9c2 cells," Toxicon, vol. 207, pp. 21-30, 2022/02/01/ 2022.
I. M. Ibrahim, D. H. Abdelmalek, and A. A. Elfiky, "GRP78: A cell's response to stress," Life Sciences, vol. 226, pp. 156-163, 2019/06/01/ 2019.
T. Verfaillie, A. D. Garg, and P. Agostinis, "Targeting ER stress induced apoptosis and inflammation in cancer," Cancer Letters, vol. 332, no. 2, pp. 249-264, 2013/05/28/ 2013.
M. Zhong, Z. Wu, Z. Chen, Q. Ren, and J. Zhou, "Advances in the interaction between endoplasmic reticulum stress and osteoporosis," Biomedicine & Pharmacotherapy, vol. 165, p. 115134, 2023/09/01/ 2023.
Z. Jin and W. S. El-Deiry, "Overview of cell death signaling pathways," Cancer Biology & Therapy, vol. 4, no. 2, pp. 147-171, 2005/02/02 2005.
T. Anelli and R. Sitia, "Protein quality control in the early secretory pathway," The EMBO Journal, vol. 27, no. 2, pp. 315-327, 2008/01/23 2008.
A. S. Lee, "The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress," (in eng), Methods, vol. 35, no. 4, pp. 373-81, 2005.
N. T. Sprenkle, S. G. Sims, C. L. Sánchez, and G. P. Meares, "Endoplasmic reticulum stress and inflammation in the central nervous system," Molecular Neurodegeneration, vol. 12, no. 1, p. 42, 2017/05/25 2017.
S. Chakravarthi, C. E. Jessop, and N. J. Bulleid, "The role of glutathione in disulphide bond formation and endoplasmic‐reticulum‐generated oxidative stress," EMBO reports, vol. 7, no. 3, pp. 271-275-275, 2006/03/01 2006.
I. B. Perez and P. J. Brown, "The role of ROS signaling in cross-tolerance: from model to crop," (in English), Frontiers in Plant Science, Mini Review vol. 5, 2014-December-23 2014.
Y. Zhang, S. Choksi, K. Chen, Y. Pobezinskaya, I. Linnoila, and Z.-G. Liu, "ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages," Cell Research, vol. 23, no. 7, pp. 898-914, 2013.
Y. Che, Y. Tian, R. Chen, L. Xia, F. Liu, and Z. Su, "IL-22 ameliorated cardiomyocyte apoptosis in cardiac ischemia/reperfusion injury by blocking mitochondrial membrane potential decrease, inhibiting ROS and cytochrome C," Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, vol. 1867, no. 9, 2021.
Q. Zhang et al., "Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress," Apoptosis, vol. 21, no. 4, pp. 432-442, 2016/04/01 2016.
Y. Tian, L. Wang, Z. Qiu, Y. Xu, and R. Hua, "Autophagy triggers endoplasmic reticulum stress and C/EBP homologous protein-mediated apoptosis in OGD/R-treated neurons in a caspase-12-independent manner," (in eng), Journal of neurophysiology, vol. 126, no. 5, pp. 1740-1750, 2021/11// 2021.
H. Wootz, I. Hansson, L. Korhonen, U. Näpänkangas, and D. Lindholm, "Caspase-12 cleavage and increased oxidative stress during motoneuron degeneration in transgenic mouse model of ALS," Biochemical and Biophysical Research Communications, vol. 322, no. 1, pp. 281-286, 2004/09/10/ 2004.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Fisetin inhibits patulin-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Materials and Reagents

2.2 The contents of LDH, SOD and MDA in cells were detected

2.3 H9c2 cardiomyocytes ROS content determination

2.4 Mitochondrial membrane potential (MMP) determination

2.5 Western blot analysis

2.6 Statistical analysis

3. Results

3.1 FIS inhibits oxidative stress induced by PAT

3.2 FIS pretreatment reduced PAT-induced ROS levels

3.3 The effect of PAT on mitochondrial membrane potential of cardiomyocytes decreased after FIS treatment

3.4 FIS inhibits PAT-induced cardiomyocyte apoptosis by regulating Grp78/Chop/Caspase-12

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1