运脾化痰通窍方下调糖酵解调控小胶质细胞极化表型改善OSA小鼠炎症及认知障碍

Yunpi Huatan Tongqiao Prescription Regulates Microglial Cell Polarization Phenotype to Improve Inflammation and Cognitive Impairment in OSA Mice by Down-regulating Glycolysis

摘要:

目的 验证运脾化痰通窍方（YHTP）下调糖酵解调控小胶质细胞表型改善阻塞性睡眠呼吸暂停（OSA）小鼠炎症及认知记忆障碍。方法 将48只雄性Balb/C小鼠随机分为正常组、模型组、孟鲁司特钠组（30 mg·kg^-1）、YHTP低、中、高剂量组（8.28、16.56、33.12 g·kg^-1），每组8只。除正常组外其他组予脂多糖（LPS）腹腔注射和慢性间歇性缺氧（CIH）造模4周，随后药物治疗4周后取材。动物行为学实验评估小鼠缺氧记忆损伤，实时荧光定量聚合酶链式反应（Real-time PCR）检测各海马组织M1相关炎症因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）及标志物T淋巴细胞活化抗原（CD86）、诱导型一氧化氮合酶（iNOS）和M2相关炎症因子白细胞介素-10（IL-10）、转化生长因子-β（TGF-β）及标志物甘露糖受体（CD206） mRNA表达水平差异。蛋白免疫印迹法（Western blot）检测海马组织M1、M2小胶质细胞表型标志物（CD86、CD206）及糖酵解相关蛋白葡萄糖转运蛋白1型（GLUT1）、己糖激酶2（HK2）、磷酸果糖激酶（PFKM）、丙酮酸激酶2（PKM2）、单羧酸转运体1（MCT1）的表达。结果 在行为学表现上，与正常组比较，模型组Y迷宫自主交替率显著降低（P<0.01），巴恩斯迷宫训练期（2、3、4 d）（P<0.05，P<0.01）及测试期（5 d、12 d）目标洞潜伏时间显著升高（P<0.01），M1胶质细胞相关标志物CD86、iNOS，炎症因子IL-1β、TNF-α mRNA显著升高（P<0.01），M2胶质细胞相关标志物IL-10、CD206、TGF-β mRNA表达显著降低（P<0.01），糖酵解蛋白HK-2、PFKM、PKM2、MCT1及M1相关标志物CD86蛋白表达显著升高（P<0.05，P<0.01），M2相关标志物CD206蛋白表达显著降低（P<0.01）。与模型组比较，YHTP中、高剂量组Y迷宫自主交替率明显升高（P<0.05，P<0.01）。训练期4 d、测试期（5、12 d）目标洞潜伏期时间显著降低（P<0.01）。Real-time PCR结果显示YHTP低、中、高剂量组小鼠海马组织M1相关促炎因子mRNA表达显著降低（P<0.01），M2相关炎症因子mRNA表达显著升高（P<0.01），Western blot结果显示YHTP中、高剂量组小鼠海马M1标志物CD86表达降低且M2标志物CD206表达显著升高（P<0.01），且糖酵解相关蛋白表达显著减少（P<0.01）。结论 YHTP可改善OSA模型小鼠炎症及缺氧认知障碍，是通过下调脑内小胶质细胞糖酵解水平，抑制细胞向M1活化，降低促炎因子释放，促进胶质细胞M2型活化，从而对OSA的炎症及认知障碍发挥治疗作用。

Abstract:

Objective To validate the efficacy of Yunpi Huatan Tongqiao prescription （YHTP） in down-regulating glycolysis to modulate microglia phenotype and improve inflammation and cognitive memory deficits in obstructive sleep apnea （OSA） mice.Method Forty-eight male Balb/C mice were randomly divided into a normal group， a model group， a montelukast sodium group （30 mg·kg^-1）， and low， medium， and high dose groups of YHTP （8.28， 16.56， and 33.12 g·kg^-1）， with 8 mice in each group. All groups， except the normal group， received intraperitoneal injections of lipopolysaccharide （LPS） and underwent chronic intermittent hypoxia （CIH） modeling for 4 weeks. Subsequently， the mice were treated with medications for 4 weeks and then sampled. Animal behavioral tests assessed memory impairment due to hypoxia. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure mRNA expression levels of M1-associated inflammatory factors interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and markers such as T lymphocyte activation antigen （CD86） and inducible nitric oxide synthase （iNOS）， as well as M2-associated inflammatory factors interleukin-10 （IL-10）， transforming growth factor-β （TGF-β）， and the marker mannose receptor （CD206） in hippocampal tissue. Western blot was employed to detect differences in the expression of M1 and M2 microglia phenotypic markers （CD86， CD206） and glycolysis-related proteins glucose transporter type 1 （GLUT1）， hexokinase 2 （HK2）， phosphofructokinase （PFKM）， pyruvate kinase 2 （PKM2）， and monocarboxylic acid transporter 1 （MCT1）.Result Behavioral tests showed that compared to the results in the normal group， the Y-maze autonomous alternation rate was significantly reduced in the model group （P<0.01）. The latency time for the target hole in the Barnes' maze during the training period （days 2， 3， 4） and testing period （days 5， 12） was significantly increased （P<0.05， P<0.01）. M1 glial cell markers CD86 and iNOS， as well as inflammatory factors IL-1β and TNF-α mRNA， were significantly elevated （P<0.01）. In contrast， the mRNA expression of M2 glial cell markers IL-10， CD206， and TGF-β was significantly reduced （P<0.01）. The protein expression of glycolytic proteins HK2， PFKM， PKM2， MCT1， and the M1 marker CD86 was significantly increased （P<0.05， P<0.01）， while M2 marker CD206 protein expression was significantly decreased （P<0.01）. Compared to the results in the model group， the Y-maze autonomous alternation rate was significantly increased in the medium and high dose groups of YHTP （P<0.05， P<0.01）. The latency time for the target hole during the training （day 4） and testing periods （days 5， 12） was significantly reduced （P<0.01）. Real-time PCR results indicated that mRNA expression levels of M1-related pro-inflammatory factors in the hippocampal tissue were significantly reduced in the low， medium， and high dose groups of YHTP （P<0.01）， while M2-related inflammatory factors' mRNA expression was significantly increased （P<0.01）. Western blot results showed that in the medium and high dose groups of YHTP， the expression of the M1 marker CD86 in the hippocampus was reduced， whereas the expression of the M2 marker CD206 was significantly increased （P<0.01）， with a significant decrease in the expression of glycolysis-related proteins （P<0.01）.Conclusion YHTP can improve inflammation and cognitive impairment induced by hypoxia in OSA model mice. This is achieved by downregulating glycolysis in brain microglia， inhibiting M1 activation， reducing pro-inflammatory factor release， and promoting M2 activation， thereby exerting a therapeutic effect on inflammation and cognitive impairment caused by OSA.