Yip1 domain family member 3/4-mediated Golgiphagy alleviates lipopolysaccharide-induced T lymphocyte apoptosis

Cheng Xinyue; Fan Qi; Chen Yu; Deng Bihan; Shi Yiqing; Han Zexin; Wu Yao; Dong Ning; Zhang Hui; Yao Yongming

doi:10.3969/j.issn.1002-1949.2025.11.003

Abstract：Objective To investigate the effect of Yip1 domain family member 3/4(YIPF3-YIPF4)-mediated Golgiphagy on lipopolysaccharide(LPS)-induced apoptosis in T lymphocytes.Methods The human T lymphocyte leukemia cell line(Jurkat)in the logarithmic growth phase was used.Cells were divided into the following groups:CON group,LPS(1 μg/mL)24 h group,LPS 48 h group,and further treated with LPS(1 μg/mL)combined with Brefeldin A(BFA,0.1 μg/mL)as the BL group or Monensin(MON,1 μg/mL)as the ML group to simulate Golgi stress conditions during sepsis(n=3).The expression of Golgi stress-related proteins(GOLPH3,GM 130)and Golgiphagy-related proteins(YIPF3,YIPF4,LC3B)was detected by Western blot.Golgi autophagy in the cells was observed by immunofluorescence.Jurkat cells with overexpression or low expression of YIPF3-YIPF4 genes were constructed by using lentiviral transfection technology.After stimulating the transfected cells with LPS for 24 h,the expression levels of apoptosis-related proteins were detected by Western blot,and the percentage of apoptotic cells was measured by flow cytometry(n=4).Results Compared with the CON group,GOLPH3 and GM130 showed the most significant changes in the LPS 24 h group(P＜0.05).Electron microscopy revealed morphological alterations in the Golgi apparatus of the LPS-stimulated group,characterized by loosely stacked Golgi membrane cisternae.In the further simulated Golgi stress experiments,compared with the CON group,the expression of YIPF3 and YIPF4 was upregulated,and the conversion of LC3 B-Ⅰ to LC3 B-Ⅱ was significantly increased in the LPS group(P＜0.05).YIPF3 expression was significantly increased in the BL group,while YIPF4 expression was significantly increased and LC3 conversion was enhanced in the ML group(P＜0.05).Immunofluorescence results showed that compared with the CON group,the expression of YIPF3 and its co-localization with the lysosomal probe were significantly enhanced in the LPS group,and this co-localization was more pronounced in the BL and ML groups.After regulating YIPF3 and YIPF4 gene expression,the protein levels of cleaved caspase-3 and Bax in the YIPF3-YIPF4 siRNA+LPS group were higher than those in the NS+LPS group(P＜0.05),while they were lower in the YIPF3-YIPF4 LV-RNA+LPS group than in the NC+LPS group(P＜0.05).Flow cytometry analysis showed that the apoptosis rate in the YIPF3-YIPF4 siRNA+LPS group was higher than that in the NS+LPS group(P＜0.05),whereas it was lower in the YIPF3-YIPF4 LV-RNA+LPS group than in the NC+LPS group(P＜0.05).Conclusion LPS stimulation induces significant Golgi stress in T lymphocytes,accompanied by the activation of YIPF3-YIPF4-mediated Golgiphagy.Promoting the expression of YIPF3-YIPF4 significantly alleviates LPS-induced apoptosis in T lymphocytes.