Malvidin induces hepatic stellate cell apoptosis via the endoplasmic reticulum stress pathway and mitochondrial pathway

Abstract Blueberries have great beneficial effects due to high level of anthocyanins, especially malvidin. Hepatic stellate cells (HSCs) can be activated and increase excessive extracellular matrix (ECM) components, which play a central role in liver fibrogenesis. Therefore, activated HSC’s apoptosis can be induced to recover liver fibrosis. Malvidin's effects on apoptosis in rat activated hepatic stellate T6 cells (HSC‐T6) in vitro were investigated here. High concentration of malvidin was found to significantly induce apoptosis, activate caspase‐3, increase malondialdehyde, upregulate Bax, but downregulate Bcl‐2. Moreover, malvidin upregulated the protein levels of some endoplasmic reticulum stress (ERS)‐typical markers, including caspase‐12, glucose‐regulated protein 78 (GRP78), and CCAAT/enhancer‐binding protein (C/EBP) homologous protein (CHOP), suggesting that malvidin induced HSC apoptosis by the ERS apoptosis pathway as well as the mitochondrial‐dependent pathway. These findings indicated that blueberry anthocyanins, especially malvidin, could induce activated hepatic stellate cell apoptosis and might act as one kind of functional food ingredient or a novel nutraceutical beneficial for liver health.

. Therefore, activated HSCs are recognized as one major source of collagenous and/or non-collagenous matrix proteins accumulated in the fibrotic liver (Gaҫa et al., 2003). Thus, the inhibiting HSC's activation or inducing activated HSCs' apoptosis provides a novel strategy for anti-fibrotic treatment (Higashi, Friedman, & Hoshida, 2017).
Anthocyanins are one type of flavonoids that widely distributed in many dietary plants, like fruits, beans, cereals, and vegetables, especially blueberries. Blueberries possess higher anthocyanin contents and stronger antioxidant activity than most other fruits and vegetables (Hosseinian & Beta, 2007;Reque et al., 2014). Many reports have confirmed that anthocyanins exhibit powerful bio-activities, including antioxidant, anti-inflammatory, anti-tumor, and hepatoprotective properties in vitro or in animal models (Mazewski, Liang, & de Mejia, 2017;Sun et al., 2018). So anthocyanins could be considered as one of the best physiologically functional phytochemicals (Prior et al., 1998).
The health benefits of anthocyanins were associated with the anthocyanin quantity via dietary intake. However, so far there is still no definite recommended daily intake of anthocyanins for optimal health in China or other countries. Recent researches have pointed out the average daily intake of anthocyanins ranged from 19.8 to 82 mg/day in different countries, involving China, Australia, and Europe (Li et al., 2013;Murphy, Walker, Dyer, & Bryan, 2019;Zamora-Ros et al., 2011). For men, the average daily anthocyanin intake has been 19.83 mg/day in Holland and 64.88 mg/day in Italy, while for women ranged from 18.73 mg/ day in Spain to 44.08 mg/day in Italy. In Finland, the consumption has already been estimated to be 82 mg/day (Morais, de Rosso, Estadella, & Pisani, 2016). The continuous research on anthocyanins will undoubtedly provide a basis for pursing healthy dietary guidance recommendations.
Our preliminary studies revealed that malvidins selectively inhibited cell proliferation in HSCs, but not in normal live cells (unpublished data). This may provide a basis for the anti-hepatic fibrosis effects of blueberry malvidins. In this study, the malvidin's effects on rat activated HSC-T6 cell apoptosis were investigated, while its possible mechanism was revealed.

| Cell viability assay
HSC-T6 cell's viability was measured by the MTT method (Zhu et al., 2012). The cells in 96-well microplate (2 × 10 4 cells/ml) were incubated with or without different Mv concentrations for 24, 48, and 72 hr. Then each well added a 20 μl of 5 mg/ml MTT to incubate for 4 hr. A 150 μl of DMSO was added after removing the supernatant, and the well was gently shaken for 10 min at 37°C. The absorbance at 490 nm was detected using a StatFax-2100 Microplate Reader (Awareness Technology Inc.). DMEM was a blank. The following formula was used to calculate: cell viability = (test OD 490 − blank OD 490 )/(control OD 490 − blank OD 490 ) × 100%.

| Fluorescence microscopy (FM) and transmission electron microscopy (TEM)
HSC-T6 cells were stained with 10 μmol/L Hoechst 33258 dye for 15 min. The stained cells were imaged using an IX53 Inverted
Samples were immediately analyzed on a Becton Dickinson (BD) FACS Calibur Flow Cytometer (BD Biosciences) at 488 and 546 nm. The early apoptotic cells were those with visible AnnexinV (green) but without PI (red) staining, while the late apoptotic cells were those only with PI staining, respectively. The percentage of apoptosis was calculated.

The relative ROS levels in the HSC-T6 cells were observed by an IX53
Inverted Fluorescent Microscope (Olympus). Fluorescent intensity was measured at the 530 nm emission wavelength and 485 nm excitation wavelength, respectively.

| Western blot
The total protein in HSC-T6 cells was detected by a BCA protein assay kit. Each 10 μg of protein was separated using 10% SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane (BIO-RAD). The primary antibodies and secondary antibodies were added orderly after blocking with 5% non-fat dry milk. The protein bands were determined by Pierce ECL substrate kit, and the levels of proteins were obtained using a LAS-3000 imaging system (Fuji) controlled by a Gel-Pro analyzer software. The untreated cell's lysate was loaded on each gel as a control. Data were expressed as the fold over the control normalized by β-actin.

| Statistical analysis
All the results were calculated as mean value of triplicate experiments ± standard deviation (SD). The figures were from Microsoft Excel 2003. Comparison between two groups was performed with t test, and comparison among multiple groups was done using analysis of variance (ANOVA). Statistical significance was acceptable when p < .05.

| Malvidin inhibited HSC-T6 cell proliferation
Different concentrations of malvidin's effects on the HSC-T6 cell proliferation were evaluated by a MTT assay. Malvidins inhibited the viability of activated HSC-T6 cells in a dose-dependent and timedependent manner ( Figure 1a). Malvidin at concentration of 50 μg/ ml could significantly decrease the cell proliferation with HSC cell viability rates down to 81.24 ± 2.66% at 24 hr and 76.22 ± 3.10% at 48 hr (both p < .05), and 70.70 ± 4.33% at 72 hr (p < .01), respectively.
As the concentration above 50 μg/ml, malvidins showed more pronounced inhibition (p < .01), and a long-time stimulation (72 hr) had a significantly stronger inhibitory effect (p < .05) than a short-time stimulation (24 hr). The cell viability rate was only 10.21 ± 1.06% for the treatment with 150 μg/ml malvidin for 72 hr.
Lactate dehydrogenase, one kind of stable cytosolic enzymes in necrotic cells, is released after membrane damaging, which is an important indicator to assess cell necrosis and could only be detected in necrotic cells (Schueren et al., 2014). Here, LDH analysis was used to determine the HSC-T6 cell necrosis and showed similar results with the MTT assay ( Figure 1b). It exhibited extremely significant difference to the control at the concentration more than 50 μg/ml (p < .01). Malvidin of all the concentrations had a significantly higher LDH activity at 72 hr than that at 24 hr (p < .01). The LDH activity of HSC-T6 cells treated with 150 μg/ml malvidin was about 3-5 folds of the control. These data suggested that malvidins induced the necrosis of HSC cells, which coincided well with the result of the MTT assay. This indicated that malvidin could induce the HSC-T6 cell apoptosis.

| Malvidin destroyed HSC-T6 cell morphology
A positive correlation between the cell apoptosis extent/damage degree and the concentration of malvidin existed.

| Malvidin triggered HSC-T6 cell ROS generation
Reactive oxygen species, recognized as a well-known inducer of apoptosis generation, can cause mitochondrial dysfunction and result in membrane depolarization (Ozben, 2007). To clarify if ROS generation involved in malvidin-induced HSC-T6 cell apoptosis, a DCFH-DA probe was applied to examine the ROS production. As shown in In addition, malondialdehyde, an oxidative damaged product of lipid peroxidation, which acts as a crucial indicator of the redox potential in cells, was also detected after incubation with malvidin for 24 hr. Results showed that the MDA content was dose-dependently upregulated in HSC-T6 cells after malvidin treatment (Figure 4b). The F I G U R E 1 Effects of malvidin on HSC-T6 cell growth. HSC-T6 cells were incubated with different concentrations of malvidin for 24, 48, or 72 hr. The cell proliferation capability was examined by MTT assay (a). The necrosis of HSC cells was measured using the LDH activity assay (b). Data are presented as mean ± SD. a p < .05, A p < .01 compared to the control at the same concentration; b p < .05, B p < .01 compared group of 24 hr with the same concentration MDA levels in cells treated with 50, 75, and 100 μg/ml Mv significantly increased from 10.86 ± 0.80 (the control) to 15.09 ± 1.12, 17.55 ± 0.67, and 21.88 ± 1.36 nmol/mg protein (p < .05). Also, a high level of malvidin (100 μg/ml) induced a significantly higher MDA level (p < .05) than a low level (50 μg/ml).
The overproduction of ROS triggers serious damages in various cells, related to increased MDA levels and decreased SOD and GSH contents. Therefore, the SOD activity and the GSH/GSSG ratio were also measured. Our results demonstrated that malvidin-induced overproduction of ROS led to a dose-dependent decrease of SOD activity and GSH/GSSG ratio (Figure 4c,d). Compared to the control, the SOD levels in cells treated with 75 and 100 μg/ml Mv significantly decreased from 203.50 ± 15.72 to 135.55 ± 12.43 and 125.33 ± 6.67 U/mg protein (p < .05), while the GSH/GSSG ratio decreased from 1.45 ± 0.06 to 0.77 ± 0.043 and 0.63 ± 0.04 (p < .05). These results provided evidence for the malvidin-induced ROS overproduction.

| Malvidin induced apoptosis through an endoplasmic reticulum stress pathway and a mitochondrial pathway
Endoplasmic reticulum stress (ERS) is important for the interplay of apoptosis in many cells (Cheng et al., 2018). Excessive ERS can induce cell apoptosis by activating critical mediators, caspase-12 and C/EBP homologous protein (CHOP; Görlach, Klappa, & Kietzmann, 2006). The endoplasmic reticulum molecular chaperone GRP78 is also connected to ERS-mediated apoptotic pathway. Considering the changes on the Malvidin downregulated the anti-apoptotic Bcl-2 level, while upregulated pro-apoptotic Bax and caspase-3 levels in HSC-T6 cells dose-dependently (Figure 5a). HSC-T6 cells with 100 μg/ml of malvidin possessed the largest changes, whose Bcl-2, Bax, and cleaved caspase-3 protein expression levels were 0.69 ± 0.03, 1.66 ± 0.08, and 1.78 ± 0.06 folds of the control (all p < .01), respectively. Bax/ Bcl-2 ratio may be more useful in determining apoptosis than each promoter. The values of the control, 50, 75, and 100 μg/ml Mv groups on the Bax/Bcl-2 ratio were 1, 1.49, 1.64, and 2.38, respectively. As shown in Figure 5b, malvidin at all the concentrations significantly increased the GRP78 and CHOP expression levels (all p < .01), which were about 1.5-2.0 folds of the control. Only a high concentration of malvidin (100 μg/ml) could significantly increase caspase-12 protein expression to 1.37 ± 0.08 folds of the control (p < .05). In addition, the 50 μg/ml Mv group had significantly different protein levels compared with the 100 μg/ml Mv group (p < .05). All these indicated that a high concentration of malvidin induced HSC-T6 cell apoptosis by an ERS pathway and a mitochondrial pathway, which might be the main reason for the decrease of HSC-T6 cells viability.

| D ISCUSS I ON
Liver fibrosis, as consequence of chronic damage in the liver, causes the extracellular matrix protein accumulation (Lee & Friedman, 2011).

F I G U R E 3
Effects of malvidin on HSC-T6 apoptosis. HSC-T6 cells were incubated with various concentrations of malvidin (Mv) for 24 hr. The apoptosis of HSC-T6 was detected using annexinV-PI double staining and flow cytometry analysis. A representative set of images from three independent experiments is shown. Results represent viable cells (Q3 quadrant), necrotic cells (Q1 quadrant), early apoptotic cells (Q4 quadrant), and late apoptotic cells (Q2 quadrant). Data are presented as mean ± SD. a p < .05, A p < .01, A* p < .001 compared to the control, b p < .05, B p < .01 versus the Mv group (50 μg/ml), c p < .05 versus the Mv group (75 μg/ml) According to recent findings, the elimination of activated HSCs and the induction of HSC apoptosis might be a crucial mechanism to terminate and potentially reverse hepatic fibrosis .
Anthocyanins from blueberries, one of important antioxidant resources, possess powerful health-promoting benefits. Many studies have proved the negative relationship between anthocyanin intake and disease risk, including cancers, chronic diseases, neurodegenerative conditions, as well as hepatic fibrosis (Braga, Murador, de Souza Mesquita, & de Rosso, 2018;Cassidy et al., 2013;Teng et al., 2017). The protective effect of anthocyanins to human body was related to the quantity of anthocyanin intake. Guo et al. (2020) reported that anthocyanin supplementation at a dose greater than F I G U R E 4 Effects of malvidin on reactive oxygen species (ROS) levels in the HSC-T6 cells. HSC-T6 cells were incubated with various concentrations of malvidin for 24 hr. Fluorescence was examined using a fluorescence microscope and levels of ROS were determined by DCFH-DA assay (a, Scale bar, 50 µm). The MDA levels (b), SOD activity (c), and the ratio of GSH/GSSG (d) were measured by corresponding test kits. Data are presented as mean ± SD. a p < .05, A p < .01 compared to the control, b p < .01, B p < .01 versus the Mv group (50 μg/ml), c p < .05 versus the Mv group (75 μg/ml) 80 mg/day is an effective agent against metabolic risk factors including oxidative and inflammatory biomarkers in healthy young adults. Barfoot et al. (2019) reported that children aged 7-10-yearold could further improve their learning and cognitive function when F I G U R E 5 Western blot analysis of apoptosis-related protein expression, including Bax, Bcl-2, Bax/Bcl-2, and cleaved caspase-3 (a) and GRP78, CHOP, and caspase-12 (b). Data are presented as mean ± SD. a p < .05, A p < .01 compared to control, b p < .05, B p < .01 versus the Mv group (50 μg/ml), C p < .01 versus the Mv group (75 μg/ml). Representative Western blots are shown they intake an anthocyanin-rich wild blueberry drink (253 mg anthocyanins). However, the dosage of anthocyanins used in the human intervention trials varies widely (100-640 mg/day) for unhealthy individuals (Qin et al., 2009;Yang et al., 2017;Zhang, Chen, Li, Ling, & Guo, 2015). High doses (≥320 mg/day) of dietary anthocyanin supplements were generally required to alleviate oxidative stress and inflammation and improve the metabolic profile in patients with chronic diseases (Li, Zhang, Liu, Sun, & Xia, 2015). The bioavailability of anthocyanin is usually very low (<1% of the initial doses; Santhakumar, Battino, & Alvarez-Suarez, 2018). Numerous studies have shown that low concentration of anthocyanins in plasma and urine has been detected (Hidalgo et al., 2012). Despite its bioavailability controversies, anthocyanin intake exhibits promising results as Reactive oxygen species have been recognized as a potential apoptosis modulator. Powerful evidences prove that cancer cells generally overproduce ROS resulting in oxidative stress (Jeelani et al., 2017). Among many ROS production resources, mitochondrion is one important and major source. The increase of mitochondrion ROS levels can induce the mitochondrial membrane potential collapse, release LDH, and increase lipid peroxidation product MDA (Zhao & Yu, 2018), which will trigger a series of mitochondria-associated events, such as activating caspase-9 (the initiator), subsequently caspase-3, caspase-6, and caspase-7 (the effector), eventually leading to apoptosis (Noh et al., 2015;Zou et al., 2018). However, the roles of ROS on mitochondrion apoptosis pathway are still unknown in Mv-induced HSC-T6 cells. In this study, ROS level, MDA concentration, and LDH release were found to increase at dose-dependent manner after Mv treatment, which was consistent with the results of apoptosis. Therefore, the oxidative stress due to ROS and MDA overproduction might be one reason for HSCs apoptosis.
Additionally, SOD is a key antioxidant enzyme, which catalyzes  (Chen et al., 2014). Thus, concentration windows should be considered when anthocyanins are used to treat liver fibrosis and cancer as pharmaceutical ingredients or nutraceuticals.
In general, the mitochondrial signaling pathway is the intrinsic pathway, which is involved in some apoptosis induced by natural products (Pan et al., 2017). Caspase cascade activation is a crucial affair in the mitochondrial apoptotic pathways. Caspase-3 is a key indicator in mitochondrial pathway-induced cell apoptosis, which play critical roles in initiating the caspase cascade reaction (Joza et al., 2008). Additionally, the proteins in Bcl-2 family are important to modulate the mitochondria-related apoptotic pathway (Chipuk et al., 2004). Bcl-2 inhibits Cyt C release, while Bax promotes the Cyt C release in the mitochondria (Elmore, 2007). The Bax/Bcl-2 ratio is used to evaluate the death or survival, which reflects maintenance of mitochondrial membrane stability after an apoptotic stimulus (Ghribi, DeWitt, Forbes, Herman, & Savory, 2001). An elevated Bax/Bcl-2 ratio indicates the cell apoptosis outbreak. In the present study, high concentrations of malvidin decreased the Bcl-2 level, while increased the Bax level and the Bax/Bcl-2 ratio. Moreover, malvidin also enhanced the expression of cleaved caspase-3, confirming the increase of cell apoptosis. The data above demonstrated that malvidin induced HSCs apoptosis through mitochondrial apoptosis pathway.
The endoplasmic reticulum (ER), one organelle important for signal transduction and protein metabolism in eukaryotic cells, contributes to the protein synthesis and transshipment in cells and serves as a pool to store calcium (Fribley, Zhang, & Kaufman, 2009).
Recent researches found the ability of ER under pressure to initiate the intrinsic pathway (Zielinski, Eigl, & Chi, 2013). However, the correlation between apoptosis and ERS has still been unknown. Caspase-12, CHOP, and GRP78 are ERS relative proteins.
Caspase-12 is an ERS-induced apoptotic marker (Nakagawa & Yuan, 2000). CHOP is the most important pro-apoptotic protein to initiate ERS-induced apoptosis (Kim, Xu, & Reed, 2008). GRP78 is dissociated with the accumulation of unfolded proteins under ERS conditions (Nakagawa & Yuan, 2000). Prolonged ERS is able to activate the caspase-12 and CHOP. In the present study, the caspase-12, CHOP, and GRP78 levels were all upregulated after malvidin treatment. These findings proved malvidin induced HSC apoptosis through the ERS-mediated pathway. That might explain that the overwhelmed ERS could greatly increase cell injury and ultimately induce cell death.

| CON CLUS IONS
Malvidin could inhibit cell proliferation and induce cell apoptosis dose-dependently in HSC-T6 cells. A high concentration of malvidin significantly evaluated ROS level and MDA concentration.
At the same time, malvidin triggered mitochondrial dysfunction by activating caspase-3, downregulating Bcl-2, and upregulating Bax. Moreover, malvidin activated endoplasmic reticulumrelated signals in HSC-T6 cells, for example, caspase-12, CHOP, and GRP78. Overall, the higher concentration of malvidin had more pronounced effects. The results indicated that malvidin could induce HSC apoptosis via the mitochondrial-dependent pathway and ERS pathway. These findings would provide a clue for blueberry anthocyanins, especially malvidin, served as a potential nutraceutical to benefit for the liver health. In the future, studies in vitro/in vivo on the functions and mechanisms of blueberry anthocyanins against hepatic fibrosis would be furtherly conducted.

CO N FLI C T O F I NTE R E S T
There is no conflict of interests regarding the publication of this paper.