Chrysophanol Ameliorates Hemin-Induced Oxidative Stress and Endoplasmic Reticulum Stress by Regulating MicroRNA-320-5p/Wnt3a Pathway in HT22 Cells

Abstract

Oxidative stress, endoplasmic reticulum (ER) stress, and neuronal cell apoptosis have been considered as the main pathogenesis factors of brain injury after intracerebral hemorrhage (ICH). Chrysophanol (CHR) has been proved to have neuroprotective effects, but the role and underlying mechanisms of CHR in ICH remain unclear. HT22 cells were dealt with hemin to mimic an in vitro ICH model and then subjected to treatment with or without CHR. The cell viability, apoptosis, ER stress, and oxidative stress were evaluated by conducting the cell counting kit-8 (CCK-8), TdT-mediated dUTP nick end labeling (TUNEL) staining assays, western blot, and corresponding kit, respectively. Further, microRNA-sequencing, bioinformatic analysis, dual-luciferase reporter method, and rescue experiments were conducted to explore the molecular mechanisms of CHR alleviating hemin-induced ER in HT22 cell. Our data revealed that CHR increased cells viability, antiapoptosis, anti-ER stress, and antioxidative stress under conditions of hemin-induced HT22 cell injury. Mechanically, it was observed that Wnt3a was competitively sponged by miR-320-5p, and CHR activated β-catenin pathway by regulating miR-320-5p/Wnt3a molecular axis. Finally, results from the rescue experiment suggested that CHR inhibited hemin-induced cells apoptosis, ER stress, and oxidative stress through regulating the miR-320-5p/Wnt3a axis in HT22 cells. In conclusion, CHR prevented hemin-induced apoptosis, ER stress, and oxidative stress via inhibiting the miR-320-5p/Wnt3a/β-catenin pathway in HT22 cells. Our results certified that CHR could be served as a promising treatment for brain damage following ICH.

Figure 1

Chrysophanol (CHR) prevented HT22 cell survival, antiapoptosis, and anti-ER stress in hemin-induced HT22 cell injury models. (a) Cell viability was studied for nonhemin (0 μM) and hemin (20 μM)-treated HT22 cells dealt with varying concentrations of CHR (0, 5, 10, 20, and 40 μM; treatment time: 24 h) by conducting CCK-8 assays. ^∗∗∗p < 0.001 vs. nonhemin. ^##p < 0.01 and ^###p < 0.001 vs. hemin. N = 3. (b) Expression of PCNA affected by CHR in hemin-induced HT22 cells was detected using the western blot. ^N.S.p ≥ 0.05 and ^∗∗p < 0.01. N = 3. (c) LDH release level was analyzed in nonhemin (0 μM) and hemin (20 μM)-treated HT22 cells in the presence and absence of CHR (0, 5, 10, 20, and 40 μM; treatment time: 24 h) by conducting the LDH cytotoxicity assay. ^N.S.p ≥ 0.05 and ^∗∗∗p < 0.001. N = 3. (d) Caspase-3 activity was studied in hemin-induced cells in the presence and absence of CHR (10 μM). ^∗∗∗p < 0.001. (e) Rate of cell apoptosis was determined using hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) by conducting TUNEL staining assays. Bar = 100 μm, ^∗∗p < 0.01. N = 3. (f) The protein expression levels of apoptosis-related genes (cleaved caspase-3, Bax, and Bcl-2) were analyzed in hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the western blot technique. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. N = 3. (g) Levels of protein expression of ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the western blot technique. ^∗p < 0.05 and ^∗∗p < 0.01. N = 3.

Figure 2

Chrysophanol (CHR) inhibited hemin-induced oxidation stress in HT22 cell. The changes of oxidation stress-related indexes (ROS, MDA, SOD, and GPx) were analyzed in the hemin-induced cells with or without CHR (10 μM) (treatment time: 24 h) using the western blot. ^∗∗∗p < 0.001. N = 3.

Figure 3

CHR inhibited HT22 cell apoptosis, ER stress, and oxidation stress by downregulating miR-320-5p in hemin-induced HT22 cell injury models. NC mim.: negative control mimics; miR mim.: miR-320-5p mimics. (a) Volcano plot generated for the miRNA expression profiles for the hemin-induced HT22 cell injury and CHR treatment groups. (b) Levels of expression of the top six miRNAs with the most significant difference in expression (identified following the sequencing method) were studied using hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the RT-PCR technique. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. N = 3. (c) Level of expression of miR-320-5p was studied using hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the RT-PCR technique. ^∗p < 0.05. N = 3. (d) The apoptosis rate in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) was studied by conducting the TUNEL staining assays. ^∗∗∗p < 0.001. N = 3. (e) Protein expression levels of the ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the western blot technique. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. N = 3. (f) The changes of oxidation stress-related indexes (ROS, MDA, SOD, and GPx) were analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h). ^∗∗p < 0.01 and ^∗∗∗p < 0.001. N = 3.

Figure 4

CHR activated the Wnt3a/β-catenin signaling pathway by downregulating miR-320-5p in hemin-induced HT22 cell injury models. (a) Potential binding site of miR-320-5p at the 3′-UTR of Wnt3a mRNA is predicted using the miRWalk software. The red color indicates wild-type, and the green color indicates the mutated sequence of Wnt3a at the miR-320-5p-binding site. (b) HT22 cells were cotransfected with miR-320-5p (or NC mimics) and a pmir-GLO vector containing wild-type or mutated miR-320-5p-binding sites (mutated) at Wnt3a 3′-UTR. Luciferase reporter assays were performed 24 h after cotransfection. NC mim.: negative control mimics; miR mim.: miR-320-5p mimics; NC inh.: negative control inhibitor; miR inh.: miR-320-5p inhibitor. ^N.S.p > 0.05, ^∗∗∗p < 0.001. N = 6. (c) HT22 cells were transfected with the miR-320-5p inhibitor or miR-320-5p mimics and their corresponding controls. The expression of miR-320-5p and Wnt3a mRNA was analyzed using the RT-PCR technique. ^∗∗∗p < 0.001. N = 3. (d and e) Expression levels of mRNA and protein of Wnt3a were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) using the RT-PCR and western blot. ^∗∗p < 0.05 and ^∗∗∗p < 0.001. N = 3. (f) Expression of miR-320-5p was analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the RT-PCR technique. ^∗∗∗p < 0.001. N = 3. (g) The mRNA expression of Wnt3a was analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the RT-PCR technique. ^∗∗∗p < 0.001. N = 3. (h) The protein expression levels of Wnt3a, total β-catenin (T-β-catenin), and nuclear β-catenin (N-β-catenin) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the western blot technique. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. N = 3.

Figure 5

CHR inhibited the cell apoptosis, ER stress, and oxidative stress by upregulating Wnt3a in hemin-induced HT22 cell injury models. Sh-NC: negative control shRNA; sh-Wnt3a: Wnt3a shRNA. (a) Protein expression levels of Wnt3a, T-β-catenin, and N-β-catenin were analyzed in hemin-induced HT22 cells (20 μM) with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) following the western blot technique. ^∗∗p < 0.01, ^∗∗∗p < 0.001. N = 3. (b) Apoptosis rate in hemin-induced HT22 cells (20 μM) with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) was studied by conducting the TUNEL staining assays. ^∗∗∗p < 0.001. N = 3. (c) The protein expression levels of ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) using the western blot technique. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. N = 3. (d) The change of oxidative stress-related indexes (MDA, SOD, GPx, and ROS) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. N = 3.