# Wild Bitter Melon Extract Abrogates Hypoxia-Induced Cell Death via the Regulation of Ferroptosis, ER Stress, and Apoptosis in Microglial BV2 Cells

Authors: Chih-Hung Lin; Jiunn-Sheng Wu; Po-Chun Hsieh; Valeria Chiu; Chou-Chin Lan; Chan-Yen Kuo
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Microglial cells are well-known phagocytic cells that are resistant to the central nervous system (CNS) and play an important role in the maintenance of CNS homeostasis. Activated microglial cells induce neuroinflammation under hypoxia and typically cause neuronal damage in CNS diseases. In this study, we propose that wild bitter melon extract (WBM) has a protective effect on hypoxia-induced cell death via regulation of ferroptosis, ER stress, and apoptosis. The results demonstrated that hypoxia caused microglial BV-2 the accumulation of lipid ROS, ferroptosis, ER stress, and apoptosis. In this study, we investigated the pharmacological effects of WBM on BV-2 cells following hypoxia-induced cell death. The results indicated that WBM reversed hypoxia-downregulated antiferroptotic molecules Gpx4 and SLC7A11, as well as upregulated the ER stress markers CHOP and Bip. Moreover, WBM alleviated hypoxia-induced apoptosis via the regulation of cleaved-caspase 3, Bax, and Bcl-2. Our results suggest that WBM may be a good candidate for preventing CNS disorders in the future.


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## Body

## 1. Introduction

Microglial cells are the main neuroimmune cells that play a central role in maintaining efficient central nervous system (CNS) homeostasis [1]. Microglial cells have also been considered the principal immune cells in the brain and are the first line of defense against the pathophysiology induced by stroke, which causes a large number of families worldwide to suffer from physical and mental burden [2]. Microglial cells are activated under ischemia and polarize into proinflammatory (M1) and anti-inflammatory (M2) phenotypes in response to stimulation associated with stroke [3]. Qin and Crews reported that activated M1-type microglia promote reactive oxygen species (ROS) overproduction via NADPH oxidase upregulation, increase the production of proinflammatory factors, and finally cause neuronal damage [4]. Therefore, targeting and promoting anti-inflammatory microglia in ischemic stroke may increase the functional recovery from neural injury and neurogenesis [5].Ferroptosis is an iron-dependent cell death, which is characterized by the accumulation of lipid ROS, downregulation of glutathione peroxidase 4 (Gpx4), and solute carrier family 7 member 11 (SLC7A11) [6]. Ferroptosis plays an increasing regulatory role in the occurrence and development of many diseases, such as neurological diseases, acute kidney injury, ischemia/reperfusion (I/R), and tumor formation [7, 8]. Accumulating evidence has shown that intracerebral hemorrhage (ICH) causes microglial cells (macrophages) to migrate into the damaged brain area to regulate the homeostasis of Fe (II) and toxic hydroxyl radicals, resulting in oxidative stress, lipid ROS overproduction, and ferroptosis [9–11]. Taken together, targeting ferroptosis in I/R-induced neural damage should consider the antiferroptosis effect to achieve optimal outcomes.Wild bitter melon (WBM;Momordica charantia L. var. abbreviata Seringe) is a wild variety of bitter melon (Momordica charantia) [12, 13]. In our previous study, we proposed that WBM has a protective effect against spinal cord injury (SCI) via a CDGSH iron sulfur domain 2 (CISD2)-dependent mechanism in SH-SY5Y human neuroblastoma cell lines and SCI mouse models [14]. Moreover, results also indicated that WBM alleviated lipopolysaccharide-induced hepatic stellate cell (HSC) activation and proliferation via the regulation of endoplasmic reticulum (ER) stress and ferroptosis [15]. Huang et al. reported that WBM exerts an anti-inflammatory effect on Propionibacterium acnes-induced skin inflammation [16]. According to a previous study, WBM could protect against cerebral ischemia/reperfusion injury by inhibiting the oxidative stress-mediated c-Jun N-terminal kinase 3 signaling pathway [17]. However, the effects of WBM on hypoxia-induced ferroptosis, ER stress, and apoptosis in microglial BV2 cells remain unclear.In this study, we propose that WBM protects against hypoxia-induced microglial BV2 cell death. Our data indicated that hypoxia decreased cell viability, increased lipid ROS accumulation, and triggered ferroptosis and ER stress via the regulation of Gpx4/SLC7A11 and CHOP/Bip in microglial BV2 cells, respectively. In this study, we verified whether attenuated hypoxia-induced microglial BV2 cell death occurs via ER stress and ferroptosis.


## 2. Materials and Methods

### 2.1. Reagents

WBM was prepared according to our previous study [14]. In brief, the extract was prepared by drying the WBM at low temperatures (20–25°C). Powdered samples of WBM were ground and stored at −20°C. Water and WBM powder were mixed at room temperature for 24 hours. To get rid of any residues, the suspension was centrifuged at 13,000 g at 4°C for 10 minutes. After freeze-drying, the supernatant was treated with ethanol at room temperature for 24 hours while it was shaken. Under vacuum, the supernatant from the centrifuged samples was concentrated and then stored at −20°C. Extracts were dissolved in absolute alcohol (Sigma-Aldrich, St. Louis, MO, USA) before analysis.


### 2.2. Cell Lines

BV2 cells were purchased and cultured in DMEM-high glucose medium containing 10% fetal bovine serum, 100°IU/mL penicillin, and 100 mg/mL streptomycin. The cells were incubated at 5% CO2 in a 37°C incubator. In this study, cell culture was performed at passages 3–10.


### 2.3. Lipid ROS Detection

Cells were incubated with 2μM C11-BODIPY 581/591 (Thermo Fisher Scientific) in culture medium for 1 h and then washed with phosphate-buffered saline. After trypsinization, the cells were collected and used for flow cytometry (BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 517–527 nm.


### 2.4. Protein Extraction and Western Blot Analysis

Protein extraction and western blot analysis were performed as previously described [18]. In brief, cell lysate was harvested in 100–120 μl of RIPA lysis buffer (#R0278, Sigma, St. Louis, MO, USA) for 30 minutes on ice. The supernatant was collected following centrifugation 10,000×g for 15 min at 4°C, and then the BCA kit (ThermoScientific Pierce BCA Protein Assay) was used to measure the protein concentration according to the manufacturer’s instructions.SDS-PAGE was performed by loading the same amount (30μg) of protein in each lane of 10% or 12% acrylamide gels. Electrophoresis was followed by membrane transfer of proteins from the gel to polyvinylidene fluoride membranes at 320 mA for 1 hour. The membrane was then blocked by soaking in 5% skim milk at room temperature for one hour while being shaken at 50–70 rpm. Primary antibodies (Gpx4, 1 : 1000 dilution, #A1933; SLC7A11, 1 : 1000 dilution, #A2413; CHOP, 1 : 1000 dilution, #A20987; Bip, 1 : 1000 dilution, #A4908; β-actin, 1 : 3000 dilution, #AC038, ABclonal, MA, USA; cleaved-caspase 3, 1 : 1000 dilution, #9661; Bax, 1 : 1000 dilution, #5023; Bcl-2, 1 : 1000 dilution, #2876, Cell Signalling, MA, USA) were then incubated at 4°C overnight to detect specific proteins on the membrane. On the following day, membranes were washed three times with TBS buffer containing 0.2% Tween 20 (Bionovas, Canada) at room temperature for 10 minutes each time. After that, membranes were incubated with a secondary antibody (ABclonal, MA, USA) diluted 1 : 5000 with horseradish peroxidase (HRP) for 1 hour. A Kodak X-ray film (USA) was used to visualize fluorescent protein signals.


### 2.5. Cell Viability Assay

Cell viability was measured using a WST-1 assay according to the manufacturer’s instructions, with some modifications in our previous study [18].


### 2.6. Statistical Analyses

All data were analyzed using one-way or two-way analysis of variance. Additionally, the Bonferroni post hoc test was used in this study. Differences were considered statistically significant when thePvalue was <0.05. ∗P<0.05and ∗∗P<0.01.


## 2.1. Reagents

WBM was prepared according to our previous study [14]. In brief, the extract was prepared by drying the WBM at low temperatures (20–25°C). Powdered samples of WBM were ground and stored at −20°C. Water and WBM powder were mixed at room temperature for 24 hours. To get rid of any residues, the suspension was centrifuged at 13,000 g at 4°C for 10 minutes. After freeze-drying, the supernatant was treated with ethanol at room temperature for 24 hours while it was shaken. Under vacuum, the supernatant from the centrifuged samples was concentrated and then stored at −20°C. Extracts were dissolved in absolute alcohol (Sigma-Aldrich, St. Louis, MO, USA) before analysis.


## 2.2. Cell Lines

BV2 cells were purchased and cultured in DMEM-high glucose medium containing 10% fetal bovine serum, 100°IU/mL penicillin, and 100 mg/mL streptomycin. The cells were incubated at 5% CO2 in a 37°C incubator. In this study, cell culture was performed at passages 3–10.


## 2.3. Lipid ROS Detection

Cells were incubated with 2μM C11-BODIPY 581/591 (Thermo Fisher Scientific) in culture medium for 1 h and then washed with phosphate-buffered saline. After trypsinization, the cells were collected and used for flow cytometry (BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 517–527 nm.


## 2.4. Protein Extraction and Western Blot Analysis

Protein extraction and western blot analysis were performed as previously described [18]. In brief, cell lysate was harvested in 100–120 μl of RIPA lysis buffer (#R0278, Sigma, St. Louis, MO, USA) for 30 minutes on ice. The supernatant was collected following centrifugation 10,000×g for 15 min at 4°C, and then the BCA kit (ThermoScientific Pierce BCA Protein Assay) was used to measure the protein concentration according to the manufacturer’s instructions.SDS-PAGE was performed by loading the same amount (30μg) of protein in each lane of 10% or 12% acrylamide gels. Electrophoresis was followed by membrane transfer of proteins from the gel to polyvinylidene fluoride membranes at 320 mA for 1 hour. The membrane was then blocked by soaking in 5% skim milk at room temperature for one hour while being shaken at 50–70 rpm. Primary antibodies (Gpx4, 1 : 1000 dilution, #A1933; SLC7A11, 1 : 1000 dilution, #A2413; CHOP, 1 : 1000 dilution, #A20987; Bip, 1 : 1000 dilution, #A4908; β-actin, 1 : 3000 dilution, #AC038, ABclonal, MA, USA; cleaved-caspase 3, 1 : 1000 dilution, #9661; Bax, 1 : 1000 dilution, #5023; Bcl-2, 1 : 1000 dilution, #2876, Cell Signalling, MA, USA) were then incubated at 4°C overnight to detect specific proteins on the membrane. On the following day, membranes were washed three times with TBS buffer containing 0.2% Tween 20 (Bionovas, Canada) at room temperature for 10 minutes each time. After that, membranes were incubated with a secondary antibody (ABclonal, MA, USA) diluted 1 : 5000 with horseradish peroxidase (HRP) for 1 hour. A Kodak X-ray film (USA) was used to visualize fluorescent protein signals.


## 2.5. Cell Viability Assay

Cell viability was measured using a WST-1 assay according to the manufacturer’s instructions, with some modifications in our previous study [18].


## 2.6. Statistical Analyses

All data were analyzed using one-way or two-way analysis of variance. Additionally, the Bonferroni post hoc test was used in this study. Differences were considered statistically significant when thePvalue was <0.05. ∗P<0.05and ∗∗P<0.01.


## 3. Results

### 3.1. ROS, ER Stress, and Ferroptosis in Hypoxia

As shown in Figure1, hypoxia caused a significant increase in lipid ROS accumulation (Figures 1(a) and 1(b)). Moreover, hypoxia significantly decreased the cell viability (Figure 1(c)). To determine whether hypoxia triggers BV-2 cell ferroptosis and ER stress, we detected the key molecules of ferroptosis and ER stress. Results showed that hypoxia significantly downregulated the antiferroptotic molecules Gpx4 and SLC7A11 (Figure 1(d)), as well as upregulated the ER stress molecules CHOP and Bip by western blotting (Figure 1(e)). Therefore, we suggest that hypoxia causes the accumulation of lipid ROS, ferroptosis, and ER stress in BV-2 cells.Figure 1
Hypoxia induces ferroptosis via the Gpx4/SLC7A11 downregulation, decreases cell survival, and induces ER stress via CHOP/Bip upregulation. (a) Changes in lipid ROS levels of control (without 2μM C11-BODIPY staining), normoxia, and hypoxia cells (with 2 μM C11-BODIPY staining). (b) Mean fluorescence intensity is used to measure lipid ROS levels. (c) WST-1 measures the viability of cells in the indicated groups. (d) The upper panel indicates expression changes of Gpx4 and SLC7A11 proteins. The lower panel indicates the quantitative results of assessing specific proteins using ImageJ. (e) The upper panel indicates the expression changes of CHOP and Bip proteins. The lower panel indicates the quantitative results of assessing specific proteins using Image =J. Three independent replicates were used to calculate the standard deviation (n = 3). ∗P<0.05. N, normoxia; H, hypoxia.
(a)(b)(c)(d)(e)


### 3.2. Hypoxic Treated with WBM

To verify whether WBM has a protective effect against hypoxia-induced cell damage, we measured the production of lipid ROS, cell viability, and expression of Gpx4, SLC7A11, CHOP, and Bip under hypoxia in the presence of WBM. The results showed that WBM significantly alleviated hypoxia-induced accumulation of lipid ROS in BV-2 cells (Figures2(a) and 2(b)). Moreover, WBM significantly attenuated hypoxia-induced cell death in a WST-1 assay. As shown in Figure 3(a), WBM significantly reversed the hypoxia-downregulated expressions of Gpx4 and SLC7A11. WBM also significantly reversed hypoxia-induced upregulation of CHOP and Bip expression (Figure 3(b)). To verify whether WBM protects BV-2 cells via ferroptosis and ER stress, we detected the expression of Gpx4 and SLC7A11 with the ferroptosis inducer erastin (20 μM) on WBM-treated BV-2 cells under hypoxia. Results indicated that erastin reversed WBM caused the upregulated Gpx4 or SLC7A11 under hypoxia conditions (Figure 3(c)). Additionally, we also detected the expression of Bio and CHOP with ER stress inducer, thapsigargin (1 μM) on WBM-treated BV-2 cells under hypoxia. Results indicated that thapsigargin (Thg) reversed WBM caused the downregulated Bio and CHOP under hypoxia condition (Figure 3(d)). Taken together, these results suggest that WBM protects against hypoxia-induced cell death via ferroptosis and ER stress in BV-2 cells.Figure 2
WBM-attenuated hypoxia induces ferroptosis and decreases cell survival. (a) Changes in lipid ROS levels of control in the absence of 2μM C11-BODIPY staining (control), hypoxia without WBM treatment in the presence of 2 μM C11-BODIPY staining (H), and hypoxia with WBM treatment in the presence of 2 μM C11-BODIPY staining (H/WBM). (b) Mean fluorescence intensity is used to measure lipid ROS levels. (c) WST-1 measures the viability of cells in the indicated groups. Three independent replicates were used to calculate the standard deviation (n = 3). ∗P<0.05.
(a)(b)(c)Figure 3
WBM-alleviated hypoxia triggers ferroptosis and ER stress in microglial BV-2 cells via Gpx4/SLC7A11 and CHOP/Bip-dependent pathways, respectively. (a) The upper panel indicates expression changes of Gpx4/SLC7A11 proteins. The lower panel indicates the quantitative results of assessing specific proteins using Image J. (b) The upper panel indicates expression changes of CHOP and Bip proteins. The lower panel indicates the quantitative results of assessing specific proteins using Image J. (c) Expression changes of Gpx4 and SLC7A11 proteins in presence or absence of 20μM erastin. (d) Expression changes of CHOP and Bip proteins in presence or absence of 1 uM thapsigargin (Thg). Three independent replicates were used to calculate the standard deviation (n = 3). ∗P<0.05. H, hypoxia. H/WBM, in presence of 30 μM WBM under hypoxia.
(a)(b)(c)(d)


### 3.3. Effect of WBM on Hypoxia-Triggered Cell Death via Apoptosis

To determine whether WBM reverses hypoxia-induced cell death via apoptosis, we detected the expressions of cleaved-caspase 3, Bax, and Bcl-2 using western blotting. The results indicated that WBM significantly attenuated the upregulation of cleaved-caspase 3 and Bax, as well as the downregulation of Bcl-2 under hypoxia (Figure4). We suggest that WBM exerts an antiapoptotic effect on hypoxia-induced cell death via apoptosis.Figure 4
WBM-alleviated hypoxia triggers apoptosis in microglial BV-2 cells via the regulation of cleaved-caspase 3, Bax, and Bcl-2. (a) The expression changes of cleaved-caspase 3, Bax, and Bcl-2 proteins. (b) The quantitative results of assessing specific proteins using ImageJ. Three independent replicates were used to calculate the standard deviation (n = 3).∗P<0.05. H, hypoxia. H/WBM, in presence of 30 μM WBM under hypoxia.
(a)(b)


## 3.1. ROS, ER Stress, and Ferroptosis in Hypoxia

As shown in Figure1, hypoxia caused a significant increase in lipid ROS accumulation (Figures 1(a) and 1(b)). Moreover, hypoxia significantly decreased the cell viability (Figure 1(c)). To determine whether hypoxia triggers BV-2 cell ferroptosis and ER stress, we detected the key molecules of ferroptosis and ER stress. Results showed that hypoxia significantly downregulated the antiferroptotic molecules Gpx4 and SLC7A11 (Figure 1(d)), as well as upregulated the ER stress molecules CHOP and Bip by western blotting (Figure 1(e)). Therefore, we suggest that hypoxia causes the accumulation of lipid ROS, ferroptosis, and ER stress in BV-2 cells.Figure 1
Hypoxia induces ferroptosis via the Gpx4/SLC7A11 downregulation, decreases cell survival, and induces ER stress via CHOP/Bip upregulation. (a) Changes in lipid ROS levels of control (without 2μM C11-BODIPY staining), normoxia, and hypoxia cells (with 2 μM C11-BODIPY staining). (b) Mean fluorescence intensity is used to measure lipid ROS levels. (c) WST-1 measures the viability of cells in the indicated groups. (d) The upper panel indicates expression changes of Gpx4 and SLC7A11 proteins. The lower panel indicates the quantitative results of assessing specific proteins using ImageJ. (e) The upper panel indicates the expression changes of CHOP and Bip proteins. The lower panel indicates the quantitative results of assessing specific proteins using Image =J. Three independent replicates were used to calculate the standard deviation (n = 3). ∗P<0.05. N, normoxia; H, hypoxia.
(a)(b)(c)(d)(e)


## 3.2. Hypoxic Treated with WBM

To verify whether WBM has a protective effect against hypoxia-induced cell damage, we measured the production of lipid ROS, cell viability, and expression of Gpx4, SLC7A11, CHOP, and Bip under hypoxia in the presence of WBM. The results showed that WBM significantly alleviated hypoxia-induced accumulation of lipid ROS in BV-2 cells (Figures2(a) and 2(b)). Moreover, WBM significantly attenuated hypoxia-induced cell death in a WST-1 assay. As shown in Figure 3(a), WBM significantly reversed the hypoxia-downregulated expressions of Gpx4 and SLC7A11. WBM also significantly reversed hypoxia-induced upregulation of CHOP and Bip expression (Figure 3(b)). To verify whether WBM protects BV-2 cells via ferroptosis and ER stress, we detected the expression of Gpx4 and SLC7A11 with the ferroptosis inducer erastin (20 μM) on WBM-treated BV-2 cells under hypoxia. Results indicated that erastin reversed WBM caused the upregulated Gpx4 or SLC7A11 under hypoxia conditions (Figure 3(c)). Additionally, we also detected the expression of Bio and CHOP with ER stress inducer, thapsigargin (1 μM) on WBM-treated BV-2 cells under hypoxia. Results indicated that thapsigargin (Thg) reversed WBM caused the downregulated Bio and CHOP under hypoxia condition (Figure 3(d)). Taken together, these results suggest that WBM protects against hypoxia-induced cell death via ferroptosis and ER stress in BV-2 cells.Figure 2
WBM-attenuated hypoxia induces ferroptosis and decreases cell survival. (a) Changes in lipid ROS levels of control in the absence of 2μM C11-BODIPY staining (control), hypoxia without WBM treatment in the presence of 2 μM C11-BODIPY staining (H), and hypoxia with WBM treatment in the presence of 2 μM C11-BODIPY staining (H/WBM). (b) Mean fluorescence intensity is used to measure lipid ROS levels. (c) WST-1 measures the viability of cells in the indicated groups. Three independent replicates were used to calculate the standard deviation (n = 3). ∗P<0.05.
(a)(b)(c)Figure 3
WBM-alleviated hypoxia triggers ferroptosis and ER stress in microglial BV-2 cells via Gpx4/SLC7A11 and CHOP/Bip-dependent pathways, respectively. (a) The upper panel indicates expression changes of Gpx4/SLC7A11 proteins. The lower panel indicates the quantitative results of assessing specific proteins using Image J. (b) The upper panel indicates expression changes of CHOP and Bip proteins. The lower panel indicates the quantitative results of assessing specific proteins using Image J. (c) Expression changes of Gpx4 and SLC7A11 proteins in presence or absence of 20μM erastin. (d) Expression changes of CHOP and Bip proteins in presence or absence of 1 uM thapsigargin (Thg). Three independent replicates were used to calculate the standard deviation (n = 3). ∗P<0.05. H, hypoxia. H/WBM, in presence of 30 μM WBM under hypoxia.
(a)(b)(c)(d)


## 3.3. Effect of WBM on Hypoxia-Triggered Cell Death via Apoptosis

To determine whether WBM reverses hypoxia-induced cell death via apoptosis, we detected the expressions of cleaved-caspase 3, Bax, and Bcl-2 using western blotting. The results indicated that WBM significantly attenuated the upregulation of cleaved-caspase 3 and Bax, as well as the downregulation of Bcl-2 under hypoxia (Figure4). We suggest that WBM exerts an antiapoptotic effect on hypoxia-induced cell death via apoptosis.Figure 4
WBM-alleviated hypoxia triggers apoptosis in microglial BV-2 cells via the regulation of cleaved-caspase 3, Bax, and Bcl-2. (a) The expression changes of cleaved-caspase 3, Bax, and Bcl-2 proteins. (b) The quantitative results of assessing specific proteins using ImageJ. Three independent replicates were used to calculate the standard deviation (n = 3).∗P<0.05. H, hypoxia. H/WBM, in presence of 30 μM WBM under hypoxia.
(a)(b)


## 4. Discussion

Increasing evidence suggests that oxygen deficiency and energy deficits during an ischemic stroke cause inflammation, oxidative stress, glutamate excitotoxicity, and apoptosis. These factors contribute to profoundly impaired neural functions. Both acute ischemic injury and the chronic recovery of brain function are affected by the inflammatory response, which leads to secondary neuronal damage. Microglia are immune cells that constantly monitor the brain microenvironment under normal circumstances [2, 19]. Therefore, hypoxia-induced neuronal damage can be effectively treated by targeting damaged microglia. Similar with our findings (Figures 1(a)–1(c), 2, and 4), Peng et al. demonstrated that propofol reduced hypoxia-induced inflammation and oxidative stress, as well as the activation of the NF-κB/Hif-1α signal under hypoxia [20]. Moreover, a previous study identified poly (ADP-ribose) polymerase-1 (PARP-1) as a previously unknown factor in amyloid β (Aβ)-induced activation of microglia, suggesting that PARP-1’s effects may be mediated, at least in part, via its interaction with NF-κB in a PARP-1 inhibitor PJ34 hAPPJ20 mouse model [21]. According to these findings, propolis inhibits the hypoxia-induced neuroinflammatory responses in microglia by inhibiting NF-κB activation [22]. Consistent with the abovementioned studies, our data indicate that WBM reduces the effects of hypoxia on microglial cell death via apoptosis, ER stress, and ROS accumulation.Developing new therapeutics for ischemic stroke that target either the proinflammatory M1 phenotype or the anti-inflammatory M2 phenotype in the future is one of the implications of current knowledge. Targeting microglia for anti-inflammatory therapy after subarachnoid hemorrhage may prevent secondary brain damage [23]. Accumulating preclinical studies have shown that these compounds or drugs have been used to alleviate stroke-induced inflammation by suppressing microglial activation or microglial cell apoptosis [24, 25]. Following traumatic brain injury (TBI), minocycline reduced chronic microglial activation, whereas a marker of neurodegeneration increased. These results indicate that microglial activation may play a role in chronic traumatic brain injury [26]. However, Yew et al. also demonstrated that taking minocycline early after stroke improved recovery, but it had a minor effect on the key features of microglial activation [27]. Therefore, the effects of minocycline on microglial activation remain unclear.Yang et al. reported that paraquat causes neuroinflammation by increasing ER stress in microglia and decreasing the proliferation of neural stem cells [28]. Interestingly, mild ER stress may protect cells from endotoxemia insults by regulating microglial polarization [29]. After arsenic exposure, microglia secreted IL-1β induced neuronal apoptosis via the PERK/eIF2α/ATF4/CHOP ER stress-mediated pathway [30] which was consistent with our results (Figures 1(e) and 3(b)). Sen et al. suggested that following TBI, aberrant ER stress induces neuronal IFNβ production and microglial activation, resulting in white matter damage caused by T-cell infiltration [31]. Accumulating evidence has demonstrated that targeting the ER in neurodegenerative diseases has potential therapeutic value [32–34].Studies have shown that iNOS/NO-enriched activated M1 macrophages/microglia are susceptible to ferroptosis but not alternatively activated M2 [35]. Research has shown that pyridoxal isonicotinoyl hydrazine suppresses ROS levels and excessive iron accumulation during hemorrhage-induced inflammation and ferroptosis, thus promoting the recovery of neurological function by regulating microglial cells [36]. Graphene quantum dots trigger ferroptosis in microglia through mitochondrial oxidative stress [37]. Based on these results, Feng et al. concluded that microglial activation after SCI caused ferroptosis of motor neurons and impaired the recovery of motor function [38]. Evidence suggests that ferroptosis is associated with multiple neurologic conditions, making it an increasingly important mechanism of CNS degeneration with microglial activation or polarization [39–41]. Consequently, targeting the proferroptosis effect on microglia in the treatment of neural diseases should consider the anti-inflammatory effect as well as the proferroptosis effect to yield optimum results [42].


## 5. Conclusion

Several findings of the existing study indicate that WBM may protect microglial BV2 cells from hypoxia-induced cell death by suppressing ferroptosis, ER stress, apoptosis, and regulation of Gpx4, SLC7A11, CHOP, and Bip (Figure5). However, to clarify the exact mechanism of its protective effect, a deep understanding of the signaling pathways and analysis of different agonists and antagonists is required. Furthermore, a comprehensive animal or clinical study is needed to prove the effectiveness of WBM in treating central nervous system diseases, including stroke, ICH, and SCI.Figure 5
The schematic depicts the functional mechanisms of WBM in hypoxia-caused damage in microglial BV-2 cells. (a) Hypoxia increased ferroptosis and apoptosis via regulations of GPX4/SLC7A11 and cleaved-caspase3/Bax/Bcl-2, respectively. Moreover, hypoxia also induced ER stress via the regulation of CHOP/Bip. (b) WBM reversed the hypoxia-triggered ferroptosis and apoptosis via regulations of GPX4/SLC7A11 and cleaved-caspase3/Bax/Bcl-2, respectively. Conversely, WBM also alleviated hypoxia-induced ER stress via the regulation of CHOP/Bip. This figure was created withhttps://www.BioRender.com.
(a)(b)



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*Source: 1072600-2022-03-16.xml*