Abstract:

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.

Key words: Traumatic brain injury, Glucose-regulated protein 75, Microglia, Neuroinflammation, Transcriptome sequencing