[Glucose-regulated protein 78 regulates endoplasmic reticulum stress in the pathogenesis of aplastic anemia]

Objective: To analyze the role of glucose-regulated protein 78 (GRP78) in regulating endoplasmic reticulum stress (ERS) in the pathogenesis of aplastic anemia (AA). Methods: The general information was collected prospectively on AA patients treated at Tianjin Medical University General Hospital from April 2022 to December 2023. According to the therapeutic effect, the newly treated AA patients were divided into the AA group, and the AA patients in remission were divided into the R-AA group. Healthy hematopoietic stem cell transplantation donors during the same period were divided into the normal control (NC) group. Bone marrow samples were collected and CD8⁺T lymphocytes were isolated to compare the expression of ERS-related genes. GRP78 protein expression level was detected and compared, and a correlation analysis with cytokines was conducted. In vitro cell experiments were conducted on bone marrow CD8⁺T lymphocytes in the AA group, which were divided into a blank group (no treatment), a tunicamycin (TM) group (10 μmol/L TM), a 4-phenylbutyrate (4-PBA) group (0.6 μmol/L 4-PBA), and a PERK inhibitor (PI) group (20 nmol/L PI). Based on different treatment concentrations, the TM group was divided into TM1 group (2 μmol/L of TM), TM2 group (4 μmol/L of TM), and TM3 group (8 μmol/L of TM). The 4-PBA group was divided into 4-PBA1 group(0.2 μmol/L of 4-PBA), 4-PBA2 group(0.4 μmol/L of 4-PBA), and 4-PBA3 group(0.8 μmol/L of 4-PBA). The PI group was divided into PI1 group(10 nmol/L of PI), PI2 group (20 nmol/L of PI), and PI3 group(30 nmol/L of PI). The expression levels of GRP78, CHOP proteins and the phosphorylation levels of p-PERK/PERK were detected and compared among the blank group, TM group, 4-PBA group, and PI group. The expression of perforin and granzyme B was compared among the subgroups of the TM group, 4-PBA group, and PI group. Results: A total of 36 patients were included, with 20 in the AA group [9 males and 11 females, aged (44±19) years], 16 in the R-AA group [10 males and 6 females, aged (35±17) years] and 18 in the NC group [4 males and 14 females, aged (44±8) years]. The mRNA expression levels of GRP78 (respectively 4.78±1.72, 1.37±0.98 and 1.04±0.67) and PERK (respectively 7.19±3.44, 3.42±1.46 and 2.47±1.15) in the AA group were higher than those in the R-AA group and the NC group (all P0.05). The expression rate of GRP78 protein in the AA group [M (Q₁, Q₃)] was higher than that in the NC group [10.30% (9.25%, 13.75%) vs 3.27% (2.41%, 4.37%), P=0.003], and the expression of GRP78 protein was positively correlated with interleukin (IL)-2 (r=0.68, P=0.021) and γ-interferon (IFN-γ) (r=0.64, P=0.036). In vitro cell experiments showed that the expression levels of GRP78 protein in the TM group were higher than those in the blank group, the expression levels of GRP78 protein in the 4-PBA group and PI group were both lower than those in the blank group. The CHOP protein expression level in the PI group was lower than that in the blank group (all P0.05). The p-PERK/PERK phosphorylation level in the PI group was lower than that in the blank group (P=0.046). The perforin expression levels in the TM1, TM2, and TM3 groups were all higher than those in the blank group, and the perforin expression level in the TM3 group was higher than that in the TM1 and TM2 groups; the expression levels of granzyme B in the TM1, TM2, and TM3 groups were all higher than those in the blank group; the expression levels of granzyme B in the TM2 and TM3 groups were higher than those in the TM1 group (all P0.05). The expression levels of perforin in the 4-PBA1, 4-PBA2, and 4-PBA3 groups were all lower than those in the blank group; the perforin expression levels in the 4-PBA3 group were lower than those in the 4-PBA1 and 4-PBA2 groups (all P0.05). The expression levels of perforin and granzyme B in the PI1, PI2, and PI3 groups were all lower than those in the blank group (P0.05). Conclusion: GRP78 regulates ERS and activates CD8⁺T lymphocytes, which may be involved in the immune pathogenesis of AA.