目的 研究白藜蘆醇通過缺氧誘導因子-1α（HIF-1α）/磷酸果糖激酶（PFK）信號軸減少小膠質(zhì)細胞糖酵解，促進大鼠脊髓損傷后的神經(jīng)修復過程的作用機制。方法 選用SD大鼠構(gòu)建繼發(fā)性脊髓損傷模型，在模型基礎上ip白藜蘆醇（25、50、100 mg/kg）干預7 d，甲苯胺藍和HE染色觀察脊髓組織中尼氏小體數(shù)量和組織病變；ELISA檢測脊髓組織炎性因子白細胞介素（IL）-1β、IL-6、腫瘤壞死因子-α（TNF-α）含量；免疫熒光染色觀察脊髓組織HIF-1α在離子鈣結(jié)合銜接分子1（IBA-1）表達和定位；蛋白質(zhì)印跡檢測脊髓組織M1標志物[誘導型一氧化氮合酶（iNOS）、CD86]、M2標志物[精氨酸酶（Arg1）、CD163]、HIF-1α、肌肉丙酮酸激酶同工酶（PKM2）、葡萄糖轉(zhuǎn)運蛋白3（GLUT3）、己糖激酶（HK）-1和乳酸脫氫酶A（LDHA）蛋白表達。構(gòu)建脂多糖（LPS）誘導小鼠小膠質(zhì)細胞（BV2）炎癥模型，在LPS（100 ng/mL）基礎上加入白藜蘆醇（30 μmol/L）和HIF-1α激動劑（DMOG，1 mmol/L）干預24 h。CCK-8檢測細胞活性，免疫熒光檢測細胞中CD86和CD163陽性表達；ELISA檢測細胞炎性因子IL-1β、IL-6、TNF-α含量；蛋白質(zhì)印跡檢測HIF-1α、PKM2、GLUT3、HK-1、LDHA的蛋白表達水平。結(jié)果 與模型組比較，白藜蘆醇組組織病變減輕，尼氏體數(shù)量增加，組織中IL-1β、IL-6、TNF-α、iNOS、CD86、HIF-1α、PKM2、GLUT3、HK-1、LDHA水平降低，Arg1、CD163水平升高（P＜0.05、0.01）；細胞結(jié)果顯示，與模型組比較，白藜蘆醇組細胞中IL-1β、IL-6、TNF-α、CD86、HIF-1α、PKM2、GLUT3、HK-1、LDHA水平降低，CD163水平升高（P＜0.01）。HIF-1α激動劑DMOG可以抑制白藜蘆醇上述效果。結(jié)論 白藜蘆醇通過抑制HIF-1α/PKM2介導的糖酵解代謝重編程，調(diào)控小膠質(zhì)細胞M2型極化傾向，進而減輕脊髓損傷后炎癥損傷。

Objective To investigate the mechanism of resveratrol promotes the nerve repair process after spinal cord injury by reducing microglia glycolysis through HIF-1α/PKM2 signaling axis.Methods Spinal cord injury model was established by using SD rats, and the rats were intraperitoneally injected with resveratrol (25, 50, and 100 mg/kg) for 7 days. Toluidine blue and HE staining were used to observe the number and pathological changes of Nissl bodies in the spinal cord tissue. ELISA was used to detect the content of IL-1β, IL-6, and TNF-α in spinal cord tissue. Immunofluorescence staining was used to observe the expression and localization of HIF-1α at IBA-1 protein. The protein expressions of M1 markers (iNOS, CD86), M2 markers (Arg1, CD163), HIF-1α, PKM2, GLUT3, HK-1, and LDHA in spinal cord tissue were detected by Western blotting. Microglia (BV2) inflammation model induced by lipopolysaccharide (LPS) was established, and resveratrol (30 mmol/L) and HIF-1α agonist (DMOG, 1 mmol/L) were added to LPS (100 ng/mL) for 24 h. CCK-8 was used to detect the cell viability, and immunofluorescence was used to detect the protein expression of CD86 and CD163 in the cells. ELISA was used to detect the levels of IL-1β, IL-6, and TNF-α. The protein expression levels of HIF-1α, PKM2, GLUT3, HK-1, and LDHA were detected by Western blotting.Results Compared with the model group, resveratrol groups alleviated the tissue lesions, increased the number of Nissl bodies, and decreased the levels of IL-1β, IL-6, TNF-α, iNOS, CD86, HIF-1α, PKM2, GLUT3, HK-1, and LDHA in the tissues, the levels of Arg-1 and CD163 were increased (P < 0.05, 0.01). Cell results showed that compared with the model group, the levels of IL-1β, IL-6, TNF-α, CD86, HIF-1α, PKM2, GLUT3, HK-1, and LDHA in the cell supernatant were decreased, but the level of CD163 was increased (P < 0.01). HIF-1α agonist DMOG could inhibit the above effects of resveratrol.Conclusions Resveratrol can regulate the M2 polarization of microglia by inhibiting HIF-1α/ PKM2-mediated glycolytic metabolic reprogramming, thereby alleviating inflammatory injury after spinal cord injury.

R285.5

延安市科技計劃項目（2023-SFGG-039）