摘要
目的探讨栝楼桂枝汤调控干扰素调控因子5(IRF5)信号通路抑制M1型小胶质细胞激活、减轻炎症反应保护受损神经细胞的分子机制。方法将小胶质细胞(BV2)分为BV2-对照组、BV2-模型组和BV2-低、中、高剂量实验组。BV2-对照组常规培养;BV2-模型组用100 ng·mL^(-1)脂多糖(LPS)刺激BV2细胞建立炎症模型,BV2-低、中、高剂量实验组分别用含50、100、200μg·mL^(-1)栝楼桂枝汤+100 ng·mL^(-1) LPS的培养基培养。将神经细胞(HT22)分为HT22-空白组、HT22-模型组、HT22-对照组和HT22-实验组,HT22-空白组常规培养;HT22-模型组进行氧糖剥夺2 h后,更换完全培养基继续培养24 h;HT22-对照组进行氧糖剥夺2 h后,更换含100 ng·mL^(-1) LPS培养基继续复氧培养24 h;HT22-实验组进行氧糖剥夺2 h后,加入200μg·mL^(-1)栝楼桂枝汤条件培养基复氧培养24 h。用酶联免疫吸附试验法检测细胞培养上清液中白细胞介素(IL)-12、IL-23含量,用蛋白质印迹法检测干扰素调控因子5(IRF5)、分化簇16(CD16)和组织相容性复合物Ⅱ(MHC-Ⅱ)的表达情况,用免疫荧光法观察神经突触标志蛋白β微管蛋白(Tuj-1)的表达情况。结果BV2-对照组、BV2-模型组和BV2-低、中、高剂量实验组的IL-12含量分别为(2.62±1.02)、(10.67±3.22)、(6.87±1.61)、(3.96±1.22)和(3.36±1.04)pg·mL^(-1),IL-23含量分别为(20.40±2.04)、(77.08±3.25)、(76.28±3.75)、(63.96±4.94)和(54.48±3.34)pg·mL^(-1),IRF5蛋白相对表达水平分别为0.80±0.41、2.22±0.69、1.11±0.11、0.92±0.39和0.65±0.29,CD16蛋白相对表达水平分别为0.69±0.45、1.91±0.52、1.42±0.22、1.04±0.15和0.67±0.30,MHC-Ⅱ蛋白相对表达水平分别为0.89±0.27、1.96±0.19、1.34±0.38、1.15±0.19和0.68±0.24。BV2-中、高剂量实验组的上述指标与BV2-模型组相比,在统计学上差异均有统计学意义(均P<0.05)。HT22-空白组、HT22-模型组、HT22-对照组和HT22-实验组的Tuj-1蛋白表达水平分别为28.85±6
Objective To explore the molecular mechanism of Gualou Guizhi decoction which regulates the interferon regulator factor 5(IRF5)signaling pathway to inhibit M1 type microglia activation and reduce the inflammatory response to protect damaged nerve cells.Methods Microglia(BV2)cells were randomly divided into BV2-control,BV2-model,BV2-experimental-L,-M,-Hgroups.The BV2-control group was given routine culture;the BV2-model group used 100 ng·mL^(-1)lipopolysaccharide(LPS)to stimulate BV2 which establish an inflammatory model;the BV2-experimental-L,-M,-H groups were cultured in 50,100,200μg·mL^(-1)GLGZD and 100 ng·mL^(-1)LPS.The HT22 cells were divided into the HT-22-blank group,HT-22-model group,HT-22-control group and HT-22 experimental group.HT-22-blank group were conventional culture;HT-22-model group were oxygen glucose deprivation was performed for 2 h,then the complete medium was replaced for 24 h;HT-22-control group were after 2 h of oxygenglucose deprivation,the 100 ng·mL^(-1)LPS conditioned medium was replaced and incubated for 24 h;HT-22-experimental group were after 2 h of oxygen glucose deprivation,the 200μg·mL^(-1)GLGZD conditioned medium was added for 24 h.Interleukin-12(IL-12)and IL-23 were detected by enzyme-linked immunosorbent assay(ELISA);the protein of IRF5,cluster differentiation 16(CD16)and MHC class Ⅱ(MHC-Ⅱ)was detected byWestern blot;the expression of the synaptic marker protein class Ⅲ β-Tubulin(Tuj-1)was observed by immunofluorescence.Results IL-12 contents in the BV2-control,BV2-model and BV2-experimental-L,-M,-H groups were(2.62±1.02),(10.67±3.22),(6.87±1.61),(3.96±1.22)and(3.36±1.04)pg·mL^(-1);IL-23 contents were(20.40±2.04),(77.08±3.25),(76.28±3.75),(63.96±4.94)and(54.48±3.34)pg·mL^(-1);relative expression levels of IRF5 protein were 0.80±0.41,2.22±0.69,1.11±0.11,0.92±0.39 and 0.65±0.29;relative expression levels of CD16 protein were 0.69±0.45,1.91±0.52,1.42±0.22,1.04±0.15 and 0.67±0.30;relative expression levels of MHC-Ⅱ protein were 0.89±0.27,1.96�
作者
钟兴华
胡海霞
林心君
朱晓勤
ZHONG Xing-hua;HU Hai-xia;LIN Xin-jun;ZHU Xiao-qin(Innovation and Transformation Center,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian Province,China;Institute of Integrated Chinese and Western Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian Province,China;Academy of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2024年第15期2197-2201,共5页
The Chinese Journal of Clinical Pharmacology
基金
福建省自然科学基金资助项目(2021J01937)。
