负载二甲双胍的甲基丙烯酰化明胶水凝胶抑制小胶质细胞炎症以促进小鼠脊髓损伤修复

Metformin-loaded gelatin methacryloyl hydrogels promote the repair by inhibiting microglial inflammation after spinal cord injury in mice

目的探究负载二甲双胍(MET)的甲基丙烯酰化明胶水凝胶(GelMA)对小鼠脊髓损伤的作用。方法将二甲双胍和小鼠小胶质细胞系(BV2)通过Transwell小室共培养,分别设置对照组,脂多糖组(LPS组)和治疗组(MET组)。共培养24 h后,通过定量聚合酶链反应(qPCR)、蛋白质印迹法(Western blot)和细胞免疫荧光技术检测不同组别的BV2细胞诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子-α(TNF-α)、CD68的表达。制备脊髓损伤小鼠横断模型,将小鼠分为假手术组(Sham组),脊髓损伤组(SCI组),单纯水凝胶修复组(GelMA组),负载二甲双胍水凝胶修复组(GelMA+MET组)。造模7天后取材进行免疫荧光实验检测iNOS、CD68的表达。造模8周内,每周进行BMS评分。采用t检验和单因素方差分析进行比较。结果MET组iNOS、TNF-α的mRNA表达(1.32±0.38、1.63±0.23)低于LPS组(2.02±0.21、4.50±0.40),差异有统计学意义(n=3,t=2.816、10.680,P<0.05)。MET组中iNOS蛋白的表达情况(0.48±0.11)低于LPS组(0.87±0.04),差异有统计学意义(n=3,t=5.641,P<0.05)。MET组的iNOS、CD68荧光强度(1.01±0.05、1.70±0.11)低于LPS组(1.40±0.07、2.49±0.07),差异有统计学意义(n=3,t=8.008、10.09,P<0.05)。GelMA+MET组的iNOS、CD68荧光数量(67.67±31.66、204.3±50.06)低于SCI组(244.0±53.67、613.0±46.13),差异有统计学意义(n=3,t=4.901、10.40,P<0.05)。第8周GelMA+MET组的BMS评分[(2.67±0.82)分]高于SCI组[(1.00±0.63)分],差异有统计学意义(n=6,t=3.953,P<0.05)。结论负载二甲双胍的GelMA通过抑制小胶质细胞炎症,减少iNOS、CD68及TNF-α的表达情况以促进小鼠脊髓损伤修复。

Objective To investigate the effect of gelatin methacryloyl hydrogels(GelMA)loaded with metformin(MET)on spinal cord injury(SCI)in mice.Methods Metformin and mice microglia cell line(BV2)were co-cultured through Transwell chamber.Control group,lipopolysaccharide(LPS)group and treatment group(MET group)were set up,respectively.After 24 h of co-culture,the expression of inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α)and CD68 in BV2 cells of different groups was detected by quantitative polymerase chain reaction(qPCR),Western blotting and immunofluorescence.The transection model of mice on spinal cord injury was prepared.The mice were divided into sham operation group(Sham group),SCI group,GelMA group,and GelMA+MET group.At 7th day after the model establishment,the expression of iNOS and CD68 was detected by immunofluorescence assay.During 8 weeks after surgery,BMS scores were performed weekly.Comparisons were made between different groups by t-test and one-way analysis of variance.Results The mRNA expression of iNOS and TNF-αin BV2 cells in the MET group(1.32±0.38,1.63±0.23)was lower than that in the LPS group(2.02±0.21,4.50±0.40),and the difference was statistically significant(n=3,t=2.816,10.680,P<0.05).The expression of iNOS protein in BV2 cells in the MET group(0.48±0.11)was lower than that in the LPS group(0.87±0.04),and the difference was statistically significant(n=3,t=5.641,P<0.05).The fluorescence intensity of iNOS and CD68 in the MET group(1.01±0.05,1.70±0.11)was lower than that in the LPS group(1.40±0.07,2.49±0.07),and the difference was statistically significant(n=3,t=8.008,10.090,P<0.05).The fluorescence amount of iNOS and CD68 in the metformin-loaded hydrogel repair group(67.67±31.66,204.3±50.06)was lower than that in the SCI group(244.0±53.67,613.0±46.13),and the difference was statistically significant(n=3,t=4.901,10.400,P<0.05).The BMS score of the metformin-loaded hydrogel repair group at 8th week(2.67±0.82)was higher than that of the SCI group(1.00±0.63),and the difference was statistically significant(n=6,t=3.953,P<0.05).Conclusion Metformin-loaded gelatin methacryloyl hydrogels promote the repair of SCI in mice by inhibiting microglial inflammation and reducing the expression of iNOS,CD68 and TNF-α.