基于CCL2-CCR2通路探讨汉黄芩素对缺氧复氧诱导神经元损伤的研究OA

Objective To investigate the effect of wogonin on hypoxia/reoxygenation(H/R)-induced neuronal injury based on the C-C chemokine ligand 2-C-C chemokine receptor 2(CCL2-CCR2)pathway.Methods PC 12 cells were cultured and divided into five groups,control group(normal culture),model group(H/R injury),experimental group(H/R+50 μmol·L-1 wogonin pretreatment for 1 h),antagonist group(H/R+10 μmol·L-1 CCR2 antagonist RS102895 pretreatment for 1 h),and agonist group(H/R+50 μmol·L-1 wogonin+20 μmol·L-1 CCL2 recombinant protein pretreatment for 1 h).Cell viability and cytotoxicity were assessed using the cell counting kit-8(CCK-8)and lactate dehydrogenase(LDH)assay kits.Apoptosis was detected by Annexin V-fluorescein isothiocyanate/propidium iodide(Annexin V-FITC/PI)double staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)staining.Oxidative stress and inflammatory responses were evaluated by enzyme-linked immunosorbent assay(ELISA)and quantitative real-time polymerase chain reaction(RT-qPCR).Protein expression levels related to the CCL2-CCR2 signaling pathway were measured by Western blot.Results The viability of PC 12 cells in control group,model group,experimental group,antagonist group and agonist group were 0.89±0.06,0.32±0.04,0.69±0.05,0.65±0.07 and 0.46±0.04,respectively;the SOD activities were(45.11±3.02),(10.05±1.05),(33.62±3.15),(30.15±2.87)and(21.05±2.17)U·mL-1,respectively;the Annexin V FITC/PI double staining apoptosis rates were(3.21±0.68)％,(29.65±1.59)％,(8.99±0.82)％,(9.87±0.85)％and(18.65±1.06)％,respectively;the TUNEL positive rates were(5.66±0.89)％,(33.65±2.42)％,(13.25±1.05)％,(11.66±1.87)％and(24.06±1.16)％,respectively;the relative expression levels of tumor necrosis factor-alpha(TNF-α)mRNA were 1.02±0.15,3.05±0.32,1.59±0.16,2.98±0.24 and 2.66±0.20,respectively;the relative expression levels of interleukin-6(IL-6)mRNA were 1.05±0.12,4.54±0.39,1.87±0.15,1.75±0.14 and 3.08±0.32,respectively;the relative expression levels of CCL2 mRNA were 1.01±0.07,3.99±0.32,1.64±0.15,3.90±0.33 and 2.84±0.23,respectively;the relative expression levels of CCR2 mRNA were 1.03±0.10,5.21±0.51,2.03±0.26,2.11±0.20 and 3.85±0.32,respectively;the relative expression levels of phosphorylation(p)p65/p65 were 0.12±0.02,0.75±0.06,0.24±0.03,0.22±0.01 and 0.52±0.05,respectively;the leakage rates of LDH were(18.32±1.15)％,(58.64±5.16)％,(29.02±2.16)％,(26.98±3.02)％and(40.24±3.54)％,respectively;the malondialdehyde(MDA)contents were(50.16±5.24),(169.65±10.16),(91.32±7.12),(86.21±5.16)and(134.16±11.87)nmol·mL-1,respectively.The differences of above indicators between model group and control group,between experimental group and model group,and between the antagonist,agonist group and experimental group were all statistically significant(all P＜0.05).Conclusion Wogonin exerts a protective effect against H/R-induced neuronal injury by inhibiting the CCL2-CCR2 signaling pathway.