Effects of curcumol on ferroptosis and tube forming ability of hepatic sinus endothelial cells

doi:10.21203/rs.3.rs-4928956/v1

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Effects of curcumol on ferroptosis and tube forming ability of hepatic sinus endothelial cells

https://doi.org/10.21203/rs.3.rs-4928956/v1

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Objective

To explore the effects of curcumol on ferroptosis and angiogenesis of hepatic sinusoidal endothelial cells, further elucidate the molecular mechanism of curcumol against liver fibrosis, and provide new ideas for the prevention and treatment of chronic liver disease.

Methods

We used VEGF to construct pathological model group, and divided hepatic sinusoidal endothelial cells into blank group, model group, high, middle and low curcumol group. Ferroptosis and angiogenesis were detected by various cell molecular biology experiments.

Results

Curcumol significantly inhibited the proliferation and migration of hepatic sinusoidal endothelial cells, significantly increased the expression of P53 and TFR1 protein, significantly decreased the expression of FTH1 protein, significantly promoted the occurrence of iron death, and significantly inhibited angiogenesis. When we knocked out p53, the effect of curcumol contributing to the onset of ferroptosis was rescued, while curcumol's role in inhibiting angiogenesis was saved, which was the same effect as when we used Ferrostatin-1.

Conclusions

Curcumol targets the P53-TFR1-FTH1 signalling axis and induces massive deposition of iron ions in hepatic sinusoidal endothelial cells, leading to the onset of ferroptosis inhibiting hepatic angiogenesis, which may be one of the molecular mechanisms of its anti-hepatic fibrosis.

curcumol

P53-TFR1-FTH1 signalling axis

ferroptosis

angiogenesis

hepatic fibrosis

Long-term stimulation of the liver by a variety of pathogenic factors can produce persistent inflammation and spontaneous over-repair of tissues, prompting hepatic stellate cells to differentiate into myofibroblasts and secrete large quantities of extracellular matrix to accumulate in the liver, leading to the development of hepatic fibrosis[1]. Liver fibrosis is a necessary pathway for chronic liver disease to progress to refractory diseases such as cirrhosis or end-stage liver disease such as hepatocellular carcinoma[2]. There is no effective anti-hepatic fibrosis treatment in modern medicine, and the complexity of the pathogenesis of liver fibrosis makes it difficult for a single-target drug to be effective, and there is a lack of definitive therapeutic drugs for the treatment of liver fibrosis. Hepatic sinusoidal endothelial cells (HSEC), as the first group of cells to contact the hepatic blood flow, play an important role in maintaining the normal physiological function of the liver and preventing the development of liver diseases[3]. Normal hepatic sinusoidal endothelial cells have a unique fenestration structure on their surface, which facilitates the efficient exchange of substances between hepatocytes and hepatic blood sinusoids[4]. In the pathological state, the hepatic sinusoids were subjected to endotoxin, ethanol and oxygen free radicals, which resulted in a reduction in the diameter and number of HSEC window holes, accompanied by the formation of subendothelial basement membranes, leading to pathological neovascularisation characterised by capillarisation of the hepatic sinusoids[5–6]. Pathological neovascularisation of the liver accompanies hepatic fibrosis, which is not only a pathological feature of hepatic fibrosis, but also one of the most important pathological reasons why hepatic fibrosis is difficult to be reversed, which occurs prior to hepatic fibrosis and continues throughout the development of hepatic fibrosis[7]. It has been shown that inhibition of pathological hepatic neovascularisation is effective in preventing the development of hepatic fibrosis[8]. Therefore, maintaining the normal phenotype of LSEC and inhibiting its pathological angiogenesis are important strategies to combat liver fibrosis. Therefore, inhibiting its pathological angiogenesis is an important strategy to combat liver fibrosis.

Ferroptosis is a new mode of programmed cell death first proposed by Dixon et al. in 2012, characterised by overloaded intracellular iron levels and oxidative damage[9]. Pathological iron accumulation can generate large amounts of reactive oxygen species (ROS) through the Fenton reaction, which reacts with polyunsaturated fatty acids on the cell membrane in an oxidative stress reaction, promoting the accumulation of toxic lipid peroxides and inducing oxidative cellular damage, leading to cellular ferroptosis[10]. Studies have shown that ROS is closely related to angiogenesis[11]. However, the relationship between ferroptosis of hepatic sinusoidal endothelial cells and angiogenesis has not been revealed. Our research group found that curcumol can regulate ferroptosis through multiple pathways and targets by means of systematic pharmacology[12]. We believe that the mechanism of action of curcumol against hepatic fibrosis has not been fully revealed. In the present study, we investigated whether curcumol affects pathological angiogenesis by mediating ferroptosis in hepatic sinusoidal endothelial cells.

2.1 Reagents and antibodies

Curcumol was obtained from Aladdin Chemical Co., Ltd (Shanghai, China); Lipofectamine ²⁰⁰⁰ transfection reagent (Life Technologies, Grand Island, NY, USA); The siRNA-targeted for P53 and a negative siRNA were designed by Gene Pharma(Shanghai, China); Ferrostatin-1 purchased from sigmma-Aldrich (Sigma, USA); The following primary antibodies against P53, TFR1 and FTH1 were purchased from Abcam (Cambridge, UK); Antibodies SLC7A11, GPX4, CD31, ACSL4 and vWF were purchased from Cell Signaling Technology (Denver, MA, USA); VEGF cytokines purchased from sigmma-Aldrich (Sigma, USA); Phosphate Buffered Saline was obtained from Solarbio(Beijing, China).

2.2 Cell culture and treatment

Mouse hepatic sinusoidal endothelial cells were purchased from procell company(Wuhan, China). Hepatic sinusoidal endothelial cells were mixed into complete medium containing 10% fetal bovine serum(Sigma, St. Louis, MO, USA), inoculated into culture flasks, and placed in an incubator at 37℃, 5% CO₂, and saturated humidity, and when the cells reached 80% confluence, the cells were cultured in 12-well plates at a density of 2×10⁵ cells per well. The cells were divided into the following five groups: blank control group, model group, curcumol 12.5mg/L group, curcumol 25 mg/L group, curcumol 50 mg/L group. The blank control group was treated only with 2% Duchenne modified eagle medium (DMEM) (Invitrogen, CA, USA); the model group was incubated with 40 ng/ml VEGF for 48 hours to activate the pathological model; and the curcumol groups were treated with 40 ng/ml VEGF for 48 hours, followed by incubation with different concentrations of curcumol for 48 hours[13].

2.3 Cell viability assay

The CCK8 kit(ab228554, Abcam, UK) was used to detect cell viability. Hepatic sinusoidal endothelial cells were inoculated into 96-well plates with 100 µL of approximately 5×10³ cells per well. After 24 hours of incubation, the cells were removed from the plates, 100 µL of CCK8 solution was added to each well, and the incubation was continued for 4 hours. The absorbance (A) value at 450 nm for each well was measured by enzyme marker(Thermo Fisher Scientific, USA).

2.4 Transwell chamber invasion assay in vitro

In 24-well plates containing serum-free media (pore diameter 8 µm; American Corning), LSEC cells were injected in equal numbers. Complete media (500 µl) was introduced to the bottom chamber in the top chamber, and the cells were fixed with paraformaldehyde(Macklin, PA, USA) and stained with crystal violet after 24 hours. Non-migrated cells in the top chamber were removed. Multiple locations in each well were randomly picked under the microscope(Olympus, Japan) for quantitative investigation.

2.5Western blot analysis

Cellular proteins were extracted, and their concentration was detected by bicinchoninic acid (BCA) (Thermo Scientific, CA, USA) with a protein marker (EpiZyme, Shanghai, China). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred to polyvinylidene fluoride (PVDF) membranes. Next, the membranes were incubated in 5% skimmed milk powder at room temperature for 1 h. Primary antibodies were then added, including SLC7A11 (1:1000), GPX4(1:1000), ACSL4 (1:1000), CD31 (1:1000), vWF (1:2000), P53(1:1000), TFR1 (1:1000), FTH1 (1:1000) and β-actin (1:1000), and incubated overnight at 4°C. The membranes were washed four times in tris-buffered saline with Tween (TBST), and incubated with a horseradish peroxidase secondary antibody (1:10000) for another hour at room temperature. The grayscale values of the target bands were analyzed using ImageJ software, and the protein expression levels were normalized by the grayscale values of the internal reference (β-actin).

2.6Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted with total RNA extraction reagent (Sigma, St. Louis, MO, USA), and primers were synthesized by Wuhan Bafeier Biotechnology Company with the help of Primer 5.0 software. According to the reverse transcription kit(Promega, USA), cDNA was synthesized. The reaction system was as follows: 95°C for 1 min; 95°C for 10 s, 60°C for 30 s, 40 cycles, with GAPDH as the internal control, and the PCR products were taken for gel imaging analysis. The relative mRNA expression level of each sample = 2^−△△Ct.The primers are shown in Table 1.

Table 1

Primer sequence list
Gene	Forward	Reverse
SLC7A11	5′-GGTCCATTACCAGCTTTTGTACG-3′	5′-AATGTAGCGTCCAAATGCCAG-3′
ACSL4	5′-CCCACTTCAGACAAACCCG-3′	5′-GTATCTGCTCCAGGGATGTCTA-3′
GPX4	5′-ATACGCTGAGTGTGGTTGC-3′	5′-CTTCATCCACTTCCACAGCG-3′
CD31	5′-GAGTCCTGCTGACCCTTCTG-3′	5′-TCAGGTTCTTCCCATTTTGC-3′
vWF	5′-GAGGCTGAGTTTGAAGTGC-3′	5′-CTGCTCCAGCTCATCCAC-3′
β-actin	5′-GCAGGAGTACGATGAGTCCG-3′	5′-CCTGACAATGACTCGACGCA-3′

2.7Immunofluorescence assay

Cells were fixed with 4% paraformaldehyde(Sinopharm Group, China) at 37°C for 60 min and then permeabilized by 0.1% Triton X-100 (Beyotime Biotech Inc,China)for 10 min. Cells were blocked with 5% bovine calf serum(Grand Island, NY, USA) for 30 min at room temperature and then incubated with indicated primary antibodies at 4°C overnight. Fluorescence was developed by incubating with Cy3 or FITC(Beyotime Biotech Inc,China) labeled goat anti-rabbit IgG(Sigma, China) for 1 h at 37°C. The nucleus was counterstained with DAPI(Beyotime Biotech Inc,China). Fluorescence image were captured under a fluorescence microscope (Japan nikon Corporation).

2.7 Prussian blue stain

Hepatic sinusoidal endothelial cells were removed from the culture medium after relevant treatment and rinsed twice with pre-warmed PBS. Fixed with 4% paraformaldehyde for 10–20 min, incubated with Prussian blue (St Louis, MO, USA) staining solution in 37°C for 30 min. Washing cells with deionized water for 2 times, re-dyeing with Prussian blue staining solution for 1 min, then re-washing it for 2–3 times. Finally, the blue particles were observed under the fluorescence microplate reader microscope (Japan Olympus Corporation).

2.8 ROS measurements

The amount of ROS in six-well cell plates was assessed using oxidation-sensitive fluorescent probe DCFH-DA (Sigma, USA) and analyzed by flow cytometry(Beckman, USA).

2.9Detection of mitochondrial membrane potential

Cells were inoculated into 6-well cell culture plates, and after the treatment of each group of cells, the cells were collected by centrifugation, and 1 mL of JC-1 (Beyotime, China) staining working solution was added to each group of cells, which were incubated at 37°C and protected from light for 30 min. After the incubation was completed, the cells were washed with PBS for 2 times and then resuspended, and detected by flow cytometry(Beckman, USA).

2.9 Observation of cell ultrastructure by transmission electron microscope

Alterations in the mitochondrial structure of hepatic sinusoidal endothelial cells observed using transmission electron microscopy(Hitachi H-7000, Japan).

2.10 Angiogenesis experiment

The 96-well plate was pre-cooled at 4°C, 150µl of Matrigel matrix gel(BD Biosciences, Bedford, MA) was added to each well and polymerised for 1 hour in a 37°C incubator and set aside, LSEC was inoculated into the wells covered with matrix gel. The angiogenesis was observed under a microscope after 8 hours of incubation and analysed using Image J software(NIH, Bethesda, MD).

2.11 Statistical analysis

IBM SPSS Statistics 25 was used for the statistical analysis (IBM, NY, USA). The means and standard deviations (SD) were used to express the data. One-way or two-way analysis of variance (ANOVA) was used to compare the groups, followed by Bonferroni post-hoc analysis. P < 0.05 was considered statistically significant.

3.1Effects of curcumol on proliferation, migration and apoptosis of HSEC

The CCK8 kit assay revealed a significant increase in HSEC proliferation rate after stimulation of HSEC with VEGF, and a significant decrease in HSEC proliferation rate when intervened with curcumol, as detailed in Fig. 1A.The Transwell assay revealed a significant increase in HSEC migratory capacity after stimulation of HSEC with VEGF, and a significant decrease in HSEC migration capacity when intervened with curcumol, as detailed in Fig. 1B. In summary, curcumol can reduce HSEC migration ability by inhibiting HSEC proliferation, thus reducing its tube-forming ability.

3.2Effect of curcumol on signal axis of P53-TFR1-FTH1 in HSEC

WB experiments revealed a significant increase in P53 and TFR1 protein expression and a significant decrease in FTH1 protein expression after VEGF stimulation of HSEC, and a significant increase in P53 and TRF1 protein expression after intervention with curcumol compared to VEGF stimulation, and a significant decrease in FTH1 protein expression compared to VEGF stimulation, as shown in detail in Fig. 2A. Immunofluorescence experiments revealed that after VEGF stimulation of HSEC P53 and TFR1 protein expression increased while FTH1 protein expression decreased, and P53 and TRF1 protein expression increased after intervention with curcumol compared to VEGF stimulation, while FTH1 protein expression decreased compared to VEGF stimulation, as detailed in Fig. 2B. Taken together, curcumol may induce lysosomal degradation of FTH1 by targeting P53 and TFR1 proteins, leading to the massive entry of iron ions into the cell.

3.3Effect of curcumol on ferroptosis of HSEC

WB and RT-PCR experiments revealed that curcumol significantly decreased the expression of SLC7A11 and GPX4, while significantly increasing the expression of ACSL4, as detailed in Fig. 3A-3B. Prussian blue staining revealed that curcumol significantly increased the deposition of ferric ions in HSEC, as detailed in Fig. 3C.ROS assay experiments revealed that curcumol could significantly ROS expression, as detailed in Fig. 3D.Transmission electron microscope Curcumol was found to significantly reduce the length of mitochondria, as detailed in Fig. 3E. In conclusion, curcumol induced ferroptosis in HSEC.

3.4Effect of curcumol on angiogenesis of HSEC

WB and RT-PCR experiments revealed a significant increase in the expression of CD31 and VWF after VEGF stimulation of HSEC and a significant decrease in the expression of CD31 and vWF when intervened with curcumol, as detailed in Figs. 4A-4B.Angiogenesis experiments revealed a significant increase in the number of neovascularisations after VEGF stimulation of HSEC and a significant decrease in the number of neovascularisations when intervened with curcumol, as detailed in see Fig. 4C. In summary, curcumol had an inhibitory effect on angiogenesis.

3.5Curcumol targets P53 to regulate ferroptosis mediated angiogenesis

To investigate the targets of curcumol's regulation of HSEC ferroptosis and to further clarify the mechanism of action of curcumol's inhibition of angiogenesis. We used si P53 and Ferropto-1, to perform functional replication experiments. Prussian blue staining revealed that curcumol-induced iron deposition was rescued by siP53, as detailed in Fig. 5A. ROS assay experiments revealed that curcumol-increased ROS expression was rescued by siP53, as detailed in Fig. 5B. Transmission electron microscopy revealed that curcumol-decreased mitochondrial length was rescued by siP53, as detailed in Fig. 5C. Mitochondrial membrane potential assay showed that the reduced mitochondrial membrane potential of curcumol could be rescued by siP53, as shown in Fig. 5D. The transwell assay showed that the inhibitory effect of curcumol on HSEC migration was rescued by siP53 or Ferrostatin-1, as shown in Fig. 5E. The angiogenesis assay revealed that the inhibition of angiogenesis by curcumol was rescued by siP53 or Ferrostatin-1, as detailed in Fig. 5F. Taken together, P53 is a key target of curcumol-induced HSEC ferroptosis, further suggesting that curcumol-induced HSEC ferroptosis is a key mechanism for its inhibition of angiogenesis.

Due to the prevalence of viral hepatitis and non-alcoholic fatty liver disease (NAFLD), liver fibrosis has become a very common disease in Asia and other parts of the world, increasing the healthcare burden on society[14]. Hepatic fibrosis occurs when the endothelial cells of the hepatic sinusoids, which are the main responding cells, continue to lose their window apertures and form a large number of contiguous basement membranes, a process known as hepatic sinusoidal capillarisation[15]. At the same time, the HSEC phenotype was greatly altered, expressing the vascular endothelial markers cluster of differentiation 31(CD31) and von Willebrand factor(vWF)[16]. Studies have shown that hepatic sinusoidal capillarisation is the main form of microvascular neovascularisation in the liver[17]. Angiogenesis plays an important role in the pathological process of liver fibrosis characterised by collagen deposition and persistent inflammatory infiltration[18]. The intimate relationship between hepatic fibrosis and neovascularisation dictates that anti-neovascularisation can be an important strategy to halt and delay the progression of hepatic fibrosis.

Ferroptosis is an iron-dependent redox imbalance that leads to cell membrane phospholipid peroxidation damage[19]. Ferroptosis was manifested morphologically as a reduction in mitochondrial volume, an increase in membrane density, a decrease or disappearance of mitochondria, rupture of the outer membrane of some mitochondria, a significant decrease in mitochondrial cristae and rupture of the cell membrane[20]. The main biochemical characteristics of ferroptosis are the accumulation of iron and lipid peroxidation, which is manifested by the Fenton reaction between excess free Fe²⁺ and hydrogen peroxide, a product of mitochondrial oxidative respiration, resulting in a large increase in reactive oxygen species (ROS), inhibition of glutathione (GSH) synthesis and glutathione peroxidase 4 (GPX4) activity, leading to impaired antioxidant system, redox imbalance, toxic lipid peroxidation, and impairment of the antioxidant system. The inhibition of GSH synthesis and GPX4 activity led to the impairment of the antioxidant system, redox imbalance, and the massive deposition of toxic lipid peroxides[21–22].

Genetically, ferroptosis is regulated by a variety of genetic factors, focusing on both lipid peroxidation and iron homeostasis[23]. The cystathionine/glutamate reverse transporter protein solute carrier family 7 member 11 (SLC7A11) promotes cystathionine uptake and glutathione biosynthesis, and its up-regulation increases intracellular cystathionine levels and accelerates glutathione biosynthesis to enhance GPX4 activity, thereby preventing oxidative stress and ferroptosis[24]. Acyl-Co A synthetase long chain family member 4 (ACSL4) plays an important role in the synthesis of polyunsaturated fatty acids, which can modulate cellular resistance to ferroptosis[25]. Studies have shown that inducing ferroptosis in human venous endothelial cells can lead to inhibition of angiogenesis[26]. Further suggesting that ferroptosis is a key upstream signal for angiogenesis[27].

P53 is a transcription factor involved in the regulation of genome integrity, cell cycle arrest, apoptosis, autophagy, altered metabolism, cellular plasticity, and the promotion of iron-dependent cell death[27–28]. P53 can reduce cystine and glutamate exchange by inhibiting SLC7A11, leading to a decrease in cysteine, the cystine reduction product of cystine in the cell, and a decrease in downstream GSH synthesis, which in turn reduces GPX4 activity, leading to the accumulation of ROS and thus inducing ferroptosis[29]. Fe²⁺ from food or erythrocyte degradation is oxidised to Fe³⁺ by plasma ceruloplasmin (CP), which binds to transferrin (TF) on the cell membrane to form TF-Fe³⁺, which enters the cell in a complex with TFR1, and is then reduced to Fe²⁺ by prostate transmembrane epithelial antigen 3 (STEAP3)[30]. Intracellular iron ions can form a ferritin complex composition with ferritin heavy chains(FTH1)[31]. Nuclear receptor coactivator 4 (NCOA4) binds to FTH1 and subsequently transports the ferritin complex to autophagic vesicles for degradation and release of iron[32]. It has been shown that up-regulation of P53 expression, increase of TRF1 expression, and down-regulation of FTH1 expression lead to massive deposition of intracellular iron ions and induce the development of ferroptosis[33]. Taken together, targeting the P53-TFR1-TFH1 signalling axis is a key strategy to regulate ferroptosis.

In recent years, the study of natural active ingredients has gradually become an important direction in the development of drugs for the treatment of diseases. Curcuma comes from a variety of plants in the genus Curcuma of the ginger family. According to Chinese medicine theory, Curcuma is warm in nature, pungent and bitter in flavour, and belongs to the liver and spleen meridians. Modern phytochemical studies show that the chemical composition of Curcuma longa mainly includes volatile oil, curcumin, polysaccharides, sterols, phenolic acids and alkaloids[34]. The main components of the volatile oil of Curcuma are monoterpenes and sesquiterpenes such as curcumol, curcumin, curcuminone, β-elemene, and curcuminone. Sesquiterpenoids possess various pharmacological activities such as antioxidant, anti-inflammatory and cytotoxic activities due to their structural specificity[35]. Curcumol is a natural product that structurally belongs to the guaiacol sesquiterpene family and has a wide range of biological activities.The anti-inflammatory and anticancer effects of curcumol have been extensively studied, and other therapeutic effects are being revealed[36–37]. We have also recently found that curcumol has significant anti-hepatic fibrosis effects in vitro and in vivo[38–39]. The above observations guided us to investigate whether the regulation of hepatic angiogenesis is related to the action of curcumol.

In our study, we found that curcumol inhibited HSEC proliferation and migration. Meanwhile, curcumol can upregulate P53 and TFR1 expression, inhibit FTH1 expression, and induce HSEC to undergo ferroptosis, but the curcumol-induced ferroptosis of HSEC can be rescued by siP53, suggesting that P53 is a key target for curcumol to regulate HSEC. Further experiments revealed that curcumol had an inhibitory effect on angiogenesis, but this effect could be rescued by si P53 or ferroptosis inhibition, suggesting that the induction of ferroptosis is a key mechanism for curcumol to inhibit angiogenesis. However, it has now also been shown that inhibition of iron death inhibits angiogenesis, which is different from our reported results[40–41].The reason for this situation is related to the different drugs and cell types, but the most important thing is the degree of ferroptosis, because the degree of iron death occurs in different levels of ROS content, ROS in a certain dose range can promote angiogenesis, if ferroptosis continues to occur in large quantities of ROS deposition, will lead to apoptosis and inhibit angiogenesis[42]. Overall, the results suggest that curcumol inhibits angiogenesis by targeting P53-TFR1-FTH1 to mediate the deposition of large amounts of iron ions in HSEC and inducing the onset of ferroptosis in HSEC, as shown in Fig. 6. These results compel us to investigate the complex mechanism between ferroptosis and angiogenesis. Of course there are some limitations in this study, such as no in vivo experiments were carried out, as well as no further clarification of the mechanism by which curcumol regulates P53 protein expression and how the increase in iron ions mediates the decrease in FTH1 protein expression.

Our study suggests that causing ferroptosis is a key strategy for regulating angiogenesis. Regulation of iron metabolism mediated by the P53-TFR1-FTH1 signalling axis is an important mechanism for regulating angiogenesis. Curcumol induced large amounts of iron ions to be deposited in HSEC by targeting the P53-TFR1-FTH1 signaling axis, contributing to the inhibition of angiogenesis by ferroptosis in HSEC. Then, from the perspective of iron ion metabolism regulating angiogenesis, we elaborated the molecular mechanism of curcumol against hepatic fibrosis, which provides a new vision and a new target for the prevention and treatment of chronic liver diseases, and also further suggests that curcumol is a lead compound for anti-hepatic fibrosis.

Consent to publish

All authors have carefully read and revised the full text and agreed to its publication.

Availability of data and materials

The data used and analyzed in this study are included within the article.

Funding

This study was funded by the National Natural Science Foundation of China (Grant No. 82204755), the Guangxi Natural Science Foundation (Grant Nos. 2023GXNSFBA026274, 2024GXNSFAA010235), the Guangxi Zhuangyao Pharmaceutical Key Laboratory (Grant No. GXZYYKF2023-05), and the Guangxi University of Traditional Chinese Medicine School-level Project Youth Fund (Grant No. 2022QN008). Faculty of Chinese Medicine Science Guangxi University of Chinese Medicine Research Project(2022MS008, 2022QJ001and 2023MS008).

Declaration of competing interest

The authors declare that there are no financial conflicts of interest.

Authors’ contributions

Jiahui Wang and Na Huang contributed equally to this work.

Acknowledgments

We thank the Guangxi University of Chinese Medicine (Guangxi, China) for providing laboratory equipment and technological support.

Higashi T, Friedman SL, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev. 2017 Nov 1;121:27-42.
Cheng D, Chai J, Wang H, Fu L, Peng S, Ni X. Hepatic macrophages: Key players in the development and progression of liver fibrosis. Liver Int. 2021 Oct;41(10):2279-2294.
Hammoutene A, Rautou PE. Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease. J Hepatol. 2019 Jun;70(6):1278-1291.
Luo X, Wang D, Zhu X, Wang G, You Y, Ning Z, Li Y, Jin S, Huang Y, Hu Y, Chen T, Meng Y, Li X. Autophagic degradation of caveolin-1 promotes liver sinusoidal endothelial cells defenestration. Cell Death Dis. 2018 May 1;9(5):576.
Xu M, Wang X, Zou Y, Zhong Y. Key role of liver sinusoidal endothelial cells in liver fibrosis. Biosci Trends. 2017 May 23;11(2):163-168.
Poisson J, Lemoinne S, Boulanger C, Durand F, Moreau R, Valla D, Rautou PE. Liver sinusoidal endothelial cells: Physiology and role in liver diseases. J Hepatol. 2017 Jan;66(1):212-227.
Elpek GÖ. Angiogenesis and liver fibrosis. World J Hepatol. 2015 Mar 27;7(3):377-391.
Zhang C, Bian M, Chen X, Jin H, Zhao S, Yang X, Shao J, Chen A, Guo Q, Zhang F, Zheng S. Oroxylin A prevents angiogenesis of LSECs in liver fibrosis via inhibition of YAP/HIF-1α signaling. J Cell Biochem. 2018 Feb;119(2):2258-2268.
Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012 May 25;149(5):1060-72.
Chen Y, Fang ZM, Yi X, Wei X, Jiang DS. The interaction between ferroptosis and inflammatory signaling pathways. Cell Death Dis. 2023 Mar 21;14(3):205.
Hsu SY, Wen ZH, Shih PC, Kuo HM, Lin SC, Liu HT, Lee YH, Wang YJ, Chen WF, Chen NF. Sinularin Induces Oxidative Stress-Mediated Apoptosis and Mitochondrial Dysfunction, and Inhibits Angiogenesis in Glioblastoma Cells. Antioxidants (Basel). 2022 Jul 23;11(8):1433.
Wang JH, He WX, Huang RJ, He JX, Li XQ, Zheng Y, Wang L. Systemic pharmacology-based exploration of the mechanism of action of curcumol in regulating ferroptosis and cellular autophagy. Acta Universitatis Medicinalis Anhui. 2022, 57(4): 621-625.
Li G, Lin J, Peng Y, Qin K, Wen L, Zhao T, Feng Q. Curcumol may reverse early and advanced liver fibrogenesis through downregulating the uPA/uPAR pathway. Phytother Res. 2020 Jun;34(6):1421-1435.
Kumar S, Duan Q, Wu R, Harris EN, Su Q. Pathophysiological communication between hepatocytes and non-parenchymal cells in liver injury from NAFLD to liver fibrosis. Adv Drug Deliv Rev. 2021 Sep;176:113869.
Li H. Intercellular crosstalk of liver sinusoidal endothelial cells in liver fibrosis, cirrhosis and hepatocellular carcinoma. Dig Liver Dis. 2022 May;54(5):598-613.
Li N, Liu C, Ma G, Tseng Y, Pan D, Chen J, Li F, Zeng X, Luo T, Chen S. Asparaginyl endopeptidase may promote liver sinusoidal endothelial cell angiogenesis via PI3K/Akt pathway. Rev Esp Enferm Dig. 2019 Mar;111(3):214-222.
Straub AC, Stolz DB, Ross MA, Hernández-Zavala A, Soucy NV, Klei LR, Barchowsky A. Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver. Hepatology. 2007 Jan;45(1):205-12.
Jiahui Wang, Xinhua Guo, Ruizhu Jiang, Jiaxi He, Tiejian Zhao, Yue Peng, Yang Zheng. Research progress in the prevention and treatment of liver fibrosis in Chinese medicine based on miRNAs molecular regulation of angiogenesis. Pharmacological Research-Modern Chinese Medicine. 2022;4:101151.
Ma XH, Liu JH, Liu CY, Sun WY, Duan WJ, Wang G, Kurihara H, He RR, Li YF, Chen Y, Shang H. ALOX15-launched PUFA-phospholipids peroxidation increases the susceptibility of ferroptosis in ischemia-induced myocardial damage. Signal Transduct Target Ther. 2022 Aug 15;7(1):288.
Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021 Apr;22(4):266-282.
Xue Q, Yan D, Chen X, Li X, Kang R, Klionsky DJ, Kroemer G, Chen X, Tang D, Liu J. Copper-dependent autophagic degradation of GPX4 drives ferroptosis. Autophagy. 2023 Jul;19(7):1982-1996.
Park E, Chung SW. ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation. Cell Death Dis. 2019 Oct 28;10(11):822.
Mou Y, Wang J, Wu J, He D, Zhang C, Duan C, Li B. Ferroptosis, a new form of cell death: opportunities and challenges in cancer. J Hematol Oncol. 2019 Mar 29;12(1):34.
Yang Y, Ma Y, Li Q, Ling Y, Zhou Y, Chu K, Xue L, Tao S. STAT6 inhibits ferroptosis and alleviates acute lung injury via regulating P53/SLC7A11 pathway. Cell Death Dis. 2022 Jun 6;13(6):530.
He S, Li R, Peng Y, Wang Z, Huang J, Meng H, Min J, Wang F, Ma Q. ACSL4 contributes to ferroptosis-mediated rhabdomyolysis in exertional heat stroke. J Cachexia Sarcopenia Muscle. 2022 Jun;13(3):1717-1730.
Zhang Y, Yang Y, Chen W, Mi C, Xu X, Shen Y, Zheng Z, Xu Z, Zhao J, Wan S, Wang X, Zhang H. BaP/BPDE suppressed endothelial cell angiogenesis to induce miscarriage by promoting MARCHF1/GPX4-mediated ferroptosis. Environ Int. 2023 Oct;180:108237.
Liu CQ, Liu XY, Ouyang PW, Liu Q, Huang XM, Xiao F, Cui YH, Zhou Q, Pan HW. Ferrostatin-1 attenuates pathological angiogenesis in oxygen-induced retinopathy via inhibition of ferroptosis. Exp Eye Res. 2023 Jan;226:109347.
Chen LL, Wang WJ. p53 regulates lipid metabolism in cancer. Int J Biol Macromol. 2021 Dec 1;192:45-54.
Nishida Xavier da Silva T, Friedmann Angeli JP, Ingold I. GPX4: old lessons, new features. Biochem Soc Trans. 2022 Jun 30;50(3):1205-1213.
Jiang L, Kon N, Li T, Wang SJ, Su T, Hibshoosh H, Baer R, Gu W. Ferroptosis as a p53-mediated activity during tumour suppression. Nature. 2015 Apr 2;520(7545):57-62.
Fang X, Ardehali H, Min J, Wang F. The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease. Nat Rev Cardiol. 2023 Jan;20(1):7-23.
Yao F, Peng J, Zhang E, Ji D, Gao Z, Tang Y, Yao X, Xia X. Pathologically high intraocular pressure disturbs normal iron homeostasis and leads to retinal ganglion cell ferroptosis in glaucoma. Cell Death Differ. 2023 Jan;30(1):69-81.
Mancias JD, Pontano Vaites L, Nissim S, Biancur DE, Kim AJ, Wang X, Liu Y, Goessling W, Kimmelman AC, Harper JW. Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis. Elife. 2015 Oct 5;4:e10308.
Bao HZ, Zhu LJ, Liu XF, Wang LJ, Luo HY. Study on the mechanism of action of Cynanchum officinale in mediating ferrotosis through up-regulation of p53 in causing liver injury in rats. Chin Tradit Herbal Drugs. 2023, 54 (02): 593-600.
Hashem S, Nisar S, Sageena G, Macha MA, Yadav SK, Krishnankutty R, Uddin S, Haris M, Bhat AA. Therapeutic Effects of Curcumol in Several Diseases; An Overview. Nutr Cancer. 2021;73(2):181-195.
Riyadi SA, Naini AA, Supratman U. Sesquiterpenoids from Meliaceae Family and Their Biological Activities. Molecules. 2023 Jun 20;28(12):4874.
Wang J, Li XM, Bai Z, Chi BX, Wei Y, Chen X. Curcumol induces cell cycle arrest in colon cancer cells via reactive oxygen species and Akt/ GSK3β/cyclin D1 pathway. J Ethnopharmacol. 2018 Jan 10;210:1-9.
Chen X, Zong C, Gao Y, Cai R, Fang L, Lu J, Liu F, Qi Y. Curcumol exhibits anti-inflammatory properties by interfering with the JNK-mediated AP-1 pathway in lipopolysaccharide-activated RAW264.7 cells. Eur J Pharmacol. 2014 Jan 15;723:339-45.
Zheng Y, Wang L, Wang JH, Liu LL, Zhao TJ. Effect of Curcumol on NOD-Like Receptor Thermoprotein Domain 3 Inflammasomes in Liver Fibrosis of Mice. Chin J Integr Med. 2022 Nov;28(11):992-999.
Zheng Y, Zhao T, Wang J, Jiang R, Huang J, Li W, Wang J. Curcumol alleviates liver fibrosis through inducing autophagy and ferroptosis in hepatic stellate cells. FASEB J. 2022;36(12):e22665.
Li G, Lin Y, Zhang Y, Gu N, Yang B, Shan S, Liu N, Ouyang J, Yang Y, Sun F, Xu H. Endometrial stromal cell ferroptosis promotes angiogenesis in endometriosis. Cell Death Discov. 2022 Jan 17;8(1):29.
Marcu R, Zheng Y, Hawkins BJ. Mitochondria and Angiogenesis. Adv Exp Med Biol. 2017;982:371-406.

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Effects of curcumol on ferroptosis and tube forming ability of hepatic sinus endothelial cells

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Abstract

Objective

Methods

Results

Conclusions

Figures

1. Introduction

2. Materials and methods

2.1 Reagents and antibodies

2.2 Cell culture and treatment

2.3 Cell viability assay

2.4 Transwell chamber invasion assay in vitro

2.5Western blot analysis

2.6Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

2.7Immunofluorescence assay

2.7 Prussian blue stain

2.8 ROS measurements

2.9Detection of mitochondrial membrane potential

2.9 Observation of cell ultrastructure by transmission electron microscope

2.10 Angiogenesis experiment

2.11 Statistical analysis

3. Results

3.1Effects of curcumol on proliferation, migration and apoptosis of HSEC

3.2Effect of curcumol on signal axis of P53-TFR1-FTH1 in HSEC

3.3Effect of curcumol on ferroptosis of HSEC

3.4Effect of curcumol on angiogenesis of HSEC

3.5Curcumol targets P53 to regulate ferroptosis mediated angiogenesis

4. Discussions

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1