Baicalin Inhibits Ferroptosis in Intracerebral Hemorrhage

Abstract

Intracerebral hemorrhage (ICH) is a subtype of stroke characterized by high mortality and disability rates. To date, the exact etiology of ICH-induced brain injury is still unclear. Moreover, there is no effective treatment to delay or prevent disease progression currently. Increasing evidence suggests that ferroptosis plays a dominant role in the pathogenesis of ICH injury. Baicalin is a main active ingredient of Chinese herbal medicine Scutellaria baicalensis. It has been reported to exhibit neuroprotective effects against ICH-induced brain injury as well as reduce iron deposition in multiple tissues. Therefore, in this study, we focused on the protective mechanisms of baicalin against ferroptosis caused by ICH using a hemin-induced in vitro model and a Type IV collagenase-induced in vivo model. Our results revealed that baicalin enhanced cell viability and suppressed ferroptosis in rat pheochromocytoma PC12 cells treated with hemin, erastin and RSL3. Importantly, baicalin showed anti-ferroptosis effect on primary cortical neurons (PCN). Furthermore, baicalin alleviated motor deficits and brain injury in ICH model mice through inhibiting ferroptosis. Additionally, baicalin existed no obvious toxicity towards the liver and kidney of mice. Evidently, ferroptosis is a key pathological feature of ICH and baicalin can prevent the development of ferroptosis in ICH. As such, baicalin is a potential therapeutic drug for ICH treatment.

Keywords: Chinese herbal medicine; baicalin; ferroptosis; hemin; intracerebral hemorrhage.

FIGURE 1

The graphical abstract of this study.

FIGURE 2

Effect of baicalin on hemin-induced ferroptosis in PC12 cells. PC12 cells were cultured in 96-well plates, treated with hemin (80μM) and corresponding concentrations of baicalin (0, 5, 10 and 20μM) for 24h. Effect of baicalin on the viability of PC12 cells was tested by MTT assay (n = 6) (A) and live/dead cell staining assay. Bar = 200μm (B). (C) The ratio of living cells was counted in different groups (n = 4). (D) The expression of GPX4 was analyzed using immunofluorescence staining. Bar = 200μm. (E) Relative fluorescence intensity statistics of the expression of GPX4 in different groups (n = 4). (F) The expression of SLC7A11 was analyzed using immunofluorescence staining. Bar = 200μm. (G) Relative fluorescence intensity statistics of the expression of SLC7A11 in different groups (n = 4). The experiments were repeated at least three times with three replicates and data were expressed as mean ± SD. ^## p < 0.01 compared with the control group and **p < 0.01 compared with the hemin-treated group.

FIGURE 3

Effect of baicalin on RSL3-induced ferroptosis in PC12 Cells. PC12 cells were cultured in 96-well plates, treated with RSL3 (0.5μM) and corresponding concentrations of baicalin (0, 5, 10 and 20μM) for 24h. Effect of baicalin on the viability of PC12 cells was tested by MTT assay (n = 6) (A) and live/dead cell staining assay. Bar = 200μm (B). (C) The ratio of living cells was counted in different groups (n = 4). (D) TEM images of mitochondrial morphology were obtained and compared between different groups. Black arrows indicate normal mitochondria, and red arrows indicate shrunken mitochondria with cristae broken or disappeared. Bar = 5μm. (E) The fluorescence intensity of DHE represents the ROS concentration in different groups. Bar = 200μm. (F) Relative fluorescence intensity statistics of the ROS concentration in different groups (n = 4). (G) The expression of GPX4 was analyzed using immunofluorescence staining. Bar = 200μm (H) Relative fluorescence intensity statistics of the expression of GPX4 in different groups (n = 4). The experiments were repeated at least three times with three replicates and data were expressed as mean ± SD. ^## p < 0.01 compared with the control group and ^* p < 0.05 and **p < 0.01 compared with the RSL3-treated group.

FIGURE 4

Effect of baicalin on erastin-induced ferroptosis in PC12 cells. PC12 cells were cultured in 96-well plates, treated with erastin (2.5μM) and corresponding concentrations of baicalin (0, 5, 10 and 20μM) for 24h. Effect of baicalin on the viability of PC12 cells was tested by MTT assay (n = 6) (A) and live/dead cell staining assay. Bar = 200μm (B). (C) The ratio of living cells was counted in different groups (n = 4). (D) TEM images of mitochondrial morphology were obtained and compared between different groups. Black arrows indicate normal mitochondria, and red arrows indicate shrunken mitochondria with cristae broken or disappeared. Bar = 5μm. (E) The fluorescence intensity of DHE represents the ROS concentration in different groups. Bar = 200μm (F) Relative fluorescence intensity statistics of the ROS concentration in different groups (n = 4) (G) The expression of SLC7A11 was analyzed using immunofluorescence staining. Bar = 200μm (H) Relative fluorescence intensity statistics of the expression of SLC7A11 in different groups (n = 4). The experiments were repeated at least three times with three replicates and data were expressed as mean ± SD. ^## p < 0.01 compared with the control group and *p < 0.05 and **p < 0.01 compared with the erastin-treated group.

FIGURE 5

Baicalin showed anti-ferroptosis effect on hemin-treated PCN (A) Representative pictures of live/dead cell staining assay and DHE assay in hemin-induced PCN treated with baicalin. Bar = 200μm (B) The ratio of living cells was counted in different groups (n = 4) (C) Relative fluorescence intensity statistics of the ROS concentration in different groups (n = 4). The experiments were repeated at least three times with three replicates and data were expressed as mean ± SD. ^## p < 0.01 compared with the control group and *p < 0.05 and **p < 0.01 compared with the hemin-treated group.

FIGURE 6

Baicalin showed anti-ferroptosis effect on erastin-treated PCN (A) Representative pictures of live/dead cell staining assay and DHE assay in erastin-induced PCN treated with baicalin. Bar = 200μm (B) The ratio of living cells was counted in different groups (n = 4) (C) Relative fluorescence intensity statistics of the ROS concentration in different groups (n = 4). The experiments were repeated at least three times with three replicates and data were expressed as mean ± SD. ^## p < 0.01 compared with the control group and *p < 0.05 and **p < 0.01 compared with the erastin-treated group.

FIGURE 7

Effect of baicalin on motor deficits in ICH model mice (n = 10–12). (A) The animal experimental flowchart. The time the mice took to turn downwards (B) and the crawling time (C) in the pole test were shown. The latency to fall (D) and total times of fall (E) in the rotarod test were shown. Experimental values were expressed as means ± SD. ^# p < 0.05 and ^## p < 0.01 compared with the sham operation group and *p < 0.05 compared with the ICH group.

FIGURE 8

Effect of baicalin on brain injury in ICH model mice. (A) Representative pictures of the hemorrhagic lesion in the mice of different groups. Bar = 0.5cm. (B) Representative images of Prussian blue staining and FJB staining of brain slices in the mice of different groups. Bar = 200μm. (C) Hematoma volume quantitative data of different groups (n = 6) (D) Relative statistics of Prussian blue labeling density in different groups (n = 4). (E) Relative fluorescence intensity statistics of FJB staining in different groups (n = 4). (F) The expression of GPX4, SLC7A11, SLC3A2, TFRC and SLC11A2 (DMT1) in the perihematoma brain tissues of mice were detected by RT-qPCR (n = 6) (G) The expression of GPX4 and SLC7A11 in the perihematoma brain tissues of ICH model mice were detected by western blot (H) Quantification data of western blot in different groups (n = 4). Experimental values were expressed as means ± SD. ^# p < 0.05 and ^## p < 0.01 compared with the sham operation group and **p < 0.01 compared with the ICH group.

FIGURE 9

Baicalin existed no obvious toxicity towards the liver and kidney. (A) Representative pictures of HE staining of liver and kidney structures in different groups (n = 4). Bar = 200μm. (B) Bar charts of serum levels of GOT, GPT and BUN in different groups (n = 6).

FIGURE 10

Proposed mechanistic model of baicalin protects against hemin-induced ferroptosis in ICH. Our findings demonstrated that baicalin effectively enhances the expression levels of GPX4 and SLC7A11, and inhibits the accumulation of lipid ROS and eventually protects against hemin-induced ferroptosis and ICH-induced brain injury.