目的

利用转录组测序技术和生物信息学分析方法研究脂多糖（LPS）对小胶质细胞系BV₂细胞基因表达水平的影响，探讨创伤性颅脑损伤（TBI）所致神经炎症中葡萄糖调节蛋白75（GRP75）的功能与作用机制。

方法

选择BV₂小胶质细胞进行实验，将细胞随机分为对照组和LPS组，对照组不做任何处理，LPS组给予1 μg/mL LPS处理6 h，每个实验分组均设置3个生物学重复，对2组细胞进行转录组测序，采用生物信息学分析方法筛选显著差异表达基因（DEGs），并对显著DEGs进行聚类分析，分析GRP75在体外细胞神经炎症模型中表达量的改变。利用定量聚合酶链式反应（qPCR）和Western blotting对GRP75的表达量进行验证。选取15只健康雄性SD大鼠，构建TBI及Sham大鼠模型，按照双随机数字表法分为假手术组（Sham组）、TBI 3 d组、TBI 7 d组、TBI 14 d组、TBI 28 d组，每组3只。利用Western blotting检测不同时段GRP75的表达情况。为研究GRP75在神经炎症中的作用机制，分别设计了GRP75过表达实验和GRP75抑制实验。在GRP75过表达实验中，将BV₂细胞转染空载质粒（Vector）和GRP75过表达质粒（GRP75-OE），分为Vector组、GRP75-OE组、Vector+LPS组和GRP75-OE+LPS组；在GRP75抑制剂处理实验中，使用5 μg/mL GRP75抑制剂MKT-077处理BV₂细胞18 h，将细胞分为CON组、MKT-077组、LPS组和LPS-MKT-077组。Western blotting检测各组细胞中诱导型一氧化氮合酶（iNOS）、环氧合酶-2（COX-2）及磷酸化核因子κB P65亚基（NF-κB P65）的蛋白水平，分析GRP75过表达和抑制对NF-κB通路的影响。

结果

转录组学质谱分析显示，LPS组中GRP75蛋白的表达水平显著低于对照组，差异有统计学意义（P＜0.05）；在BV₂细胞神经炎症模型中，LPS组的GRP75蛋白表达水平明显低于对照组，差异有统计学意义（P＜0.05）。在TBI大鼠脑组织中，与Sham组相比，TBI 3 d组、TBI 7 d组、TBI 14 d组和TBI 28 d组的GRP75蛋白相对表达量均下降，差异均有统计学意义（P＜0.05）。在GRP75发挥抑炎作用机制中，GRP75过表达实验显示，与Vector+LPS组相比，GRP75-OE+LPS组iNOS、COX-2、磷酸化NF-κB P65的表达水平均显著降低，差异有统计学意义（P＜0.05）；GRP75抑制剂处理实验显示，与LPS组相比，MKT-077+LPS组iNOS、COX-2、磷酸化NF-κB P65表达水平均上调，差异有统计学意义（P＜0.05）。

结论

GRP75在TBI大鼠脑组织及体外神经炎症模型中表达下调，GRP75过表达可显著减轻神经炎症的水平，其机制可能是通过NF-κB信号通路实现的，从而减轻TBI后的神经炎症并减少继发性损伤。因此，GRP75可能在TBI中发挥保护作用，并有望成为减轻TBI诱导的神经炎症的潜在治疗靶点。

Objective

To investigate the effects of lipopolysaccharide （LPS） on gene expression levels in the BV₂ microglial cell line using transcriptome sequencing and bioinformatics analysis， and to explore the function and mechanism of glucose-regulated protein 75 （GRP75） in neuroinflammation induced by traumatic brain injury （TBI）.

Methods

BV₂ cells were selected for the experiment and randomly divided into the control group and an LPS group. The control group did not receive any treatment， while the LPS group was treated with 1 μg/mL LPS for 6 h. Three biological replicates were set up for each experimental group. Transcriptome sequencing was performed on the two groups of cells.Bioinformatics analysis was used to screen for significantly differentially expressed genes （DEGs）， and cluster analysis was performed on the significant DEGs to analyze the changes in the expression level of GRP75 in an in vitro cellular neuroinflammation model. The expression level of GRP75 was validated using qPCR and Western blotting. Fifteen healthy male SD rats were selected to construct TBI and Sham rat models. They were divided into sham operation group （Sham group）， TBI 3 d group， TBI 7 d group， TBI 14 d group， and TBI 28 d group according to the double random number table method， with three rats in each group. Western blotting was used to detect the expression of GRP75 at different time periods. To investigate the mechanism of action of GRP75 in neuroinflammation， GRP75 overexpression experiments and GRP75 inhibition experiments were designed separately. In the GRP75 overexpression experiment，BV₂ cells were transfected with empty vector plasmid （Vector） and GRP75 overexpression plasmid（GRP75-OE）， and divided into Vector group， GRP75-OE group， Vector+LPS group， and GRP75-OE+LPS group； In the GRP75 inhibitor treatment experiment， BV₂ cells were treated with 5 μg/mL GRP75 inhibitor MKT-077 for 18 h， and the cells were divided into CON group， MKT-077 group， LPS group， and LPS-MKT-077 group. Western blotting was used to detect the protein levels of inducible nitric oxide synthase （iNOS）， cyclooxygenase-2 （COX-2）， and phosphorylated nuclear factor kappa B P65 subunit（NF-κB P65） in each group of cells， and the effects of GRP75 overexpression and inhibition on the NF-κB pathway were analyzed.

Results

Transcriptomic analysis indicated that GRP75 protein expression levels in the LPS group were significantly lower than those in the control group （P＜0.05）. Validation experiments showed that GRP75 expression in the the LPS group was significantly lower than that in the control group （P＜0.05）. In TBI rat brain tissues， compared with the Sham group， the relative expression levels of GRP75 protein decreased in the TBI 3 d group， TBI 7 d group， TBI 14 d group， and TBI 28 d group （P＜0.05）. In experiments exploring the anti-inflammatory mechanism of GRP75， overexpression experiments of GRP75 showed that iNOS， COX-2， and phosphorylated NF-κB P65 levels were significantly reduced in the GRP75-OE+LPS group compared to the Vector+LPS group （P＜0.05）. The GRP75 inhibitor treatment experiment showed that iNOS， COX-2， and phosphorylated NF-κB P65 levels were significantly increased in the MKT-077+LPS group compared to the LPS group （P＜0.05）.

Conclusions

GRP75 expression is downregulated in TBI rat brain tissues and in vitro neuroinflammation models. Overexpression of GRP75 significantly reduces neuroinflammation， potentially by modulating the NF-κB pathway， thereby alleviating neuroinflammation following TBI and reducing secondary damage. GRP75 may therefore play a protective role in TBI and serve as a potential therapeutic target for mitigating TBI-induced neuroinflammation.