Effect Of CCK-8 On Endoplasmic Reticulum Stress In LPS-induced RAW264.7 Cells

Objective: Inflammation is a protective reaction of tissue to all kinds of injured factors, but uncontrolled inflammation become an important mechanism by which infectious diseases developed multiple organ dysfunction syndromes. Actived monocyte-macrophage plays a key role in the uncontrolled inflammation. LPS(lipopolysaccharide, LPS) or other proinflammatory factors contribute to generation and release of large amounts of proinflammatory cytokines in macrophages, including IL-1β, IL-6, et al, which causes tissues damage, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome and even death. Therefore, effective control of monocyte-macrophage cells excessive activation plays an active role in reducing inflammation reaction. In eukaryotic cells, the protein is synthesized mainly in endoplasmic reticulum, and endoplasmic reticulum is very sensitive to stress stimulus. This leads to endoplasmic reticulum stress(ERS), which is defined as an imbalance between the cellular demand for ER function and ER capacity. To reduce the excessive protein loading, the cells trigger the unfolded protein response(UPR), which signals transient attenuation of protein translation, degradation of malfolded proteins and the induction of molecular chaperones and folding enzymes to augment the ER capacity of protein folding and degradation. However, if the ER stress cannot be relieved, apoptotic pathways are activated in the damaged cells.Many studies have shown that ERS plays a key role in inflammatory responses of macrophage induced by LPS. LPS attacks macrophageand cause excessive protein loading GRP78, also referred to as Bi P, is a central regulator of the UPR. Under normal conditions, GRP78 binds to the N-termini of ER-localized transmembrane proteins, including inositol-requiring enzyme 1(IRE1) When unfolded proteins induced by multiple factors accumulate in the ER lumen, GRP78 releases these transmembrane ER proteins, which activates ERS7-12, 24, then inositol required element-1(inositol requiring element-1, IRE1) is released. Activated IRE1 cuts the X-box binding protein transcription factor 1(X-box binding protein 1, XBP1) m RNA, and makes it from inactivated to activated, then translates it into XBP1 protein. The XBP1 protein acts on the endoplasmic reticulum stress response element(ER stress response element, ERSE), and promotes the transcription and expression of C/EBP homologous protein(C/EBP homologous protein, CHOP). Increased expression of CHOP can activate caspase protease-11/caspase protease-1(caspase-11/caspase-1) signaling pathway and promote the generation of IL-1β, which become an key Signal transduction pathway in inflammation. Therefore, controlling the degree of ERS is very important to the inhibition of LPS-induced inflammation. CCK(cholecystokinin, CCK) is a kind of brain-gut peptide which exists widely in various systems in vivo. It involves in the regulation of a variety of biological functions, such as nerve-humoral regulation, memory regulation and feeding regulation. CCK exists in the body in various forms, and cholecystokinin octopeptide(CCK-8), which lies in the small intestine, blood and central nervous system, is the most important form among them. Previous studies in our laboratory showed that CCK-8 had anti-inflammatory and immunomodulatory effects. For example, CCK-8 could alleviate inflammation damage in endotoxin shock rats lungs, livers, spleens and renals as well as inhibit the release of a variety of pro-inflammatory cytokines in vivo. Therefore, CCK-8 has good application prospects for the treatment of inflammation-related diseases. Based on the background above, the present study chosed stable passaged mouse macrophage cell line RAW264.7 cell to explore the effects of CCK-8 on LPS-induced ERS, which will provide a theoretical basis for clinical development and application of CCK-8 treatment for LPS-related inflammatory diseases.Methods:1 Monocyte-macrophage cell leukemia cells(cells were purchased from the Chinese Academy cell bank) RAW264.7 cells in mouse were chosen as the subjects. 2 Experimental groups: RAW264.7 cells in mouse were randomly divided into six groups: control group(Con group): cells were cultured by serum-free DMEM/high glucose culture medium; LPS group: cells were cultured by serum-free culture medium which contains 0.5μg/ml LPS; CCK-8 group: cells were cultured by serum-free culture medium which contains 10-6μmol/ml CCK-8; CCK-8+LPS group: cells were cultured by a serum-free medium treated by 10-6μmol/ml CCK-8 for 10 min at first, then mixed with LPS-treated cells; CCK-8 receptor antagonists-1+ CCK-8 + LPS group(devazepide+CCK-8 + LPS): cells were cultured by a serum-free medium that is interfused with 10-3M devazepide. 10 min before it CCK-8 and LPS were interfused; CCK-8-1 receptor antagonist group(devazepide group): cells were cultured by a serum-free medium which contains 10-3M devazepide. The dosages of the drugs and the time were in accordance with the pre-experimental results. 3 The cells were collected and total protein was extracted. The expression of endoplasmic reticulum stress marker GRP78/Bip and CHOP protein were detected by Western blot. Total RNA was extracted and RT-PCR was used to detect changes in expressions of XBP-1s m RNA in RAW264.7 cells. Cell supernatants were collected and IL-1β levels of were detected by ELISA in supernatant. 4 Data were expressed with mean±standard deviation(Mean ± SD), and statistical analysis was accomplished by SPSS21.0 software. Comparisons of means in each groups were accomplished by ANOVA analysis(ANOVA). Pairwise comparisons were accomplished by the least significant difference method(least significant difference, LSD). P<0.05 was considered statistically significant. Results: 1 The effects of different concentrations of LPS and different times oftreatment on the protein expressions of GRP78 and CHOP With the 24-hour treatment of different concentrations of LPS(0.5,1.0,1.5μg/ml) in RAW264.7 cells, expressions of GRP78 and CHOP were significantly increased compared with that of control group(P<0.05). No statistical difference was observed in the expressions of GRP78/Bip and CHOP among all LPS groups(0.5,1.0,1.5μg/ml)(P>0.05). considering the toxic effect of LPS, So 0.5μg/ml was chosen in the subsequent experiments. In RAW264.7 cells, after treated with 0.5μg/ml LPS at different times(3, 6, 12, 24h), the expression of CHOP was increased compared with the control group. Moreover, the peak was at 12h(P<0.05). The changes inexpression of CHOP and GRP78/Bip were consistent. According to the results above, treatment with 0.5μg/ml LPS for 12 h was be confirmed as final experimental condition. 2 The effect of CCK-8 on expression of XBP1-s m RNA in LPS-induced RAW264. 7 cell Elevated expression levels of XBP1-s m RNA is a signal of IRE1 activation. Compared with the control group, the expression of XBP1-s m RNA in LPS group was significantly increased. Compared with LPS group, the expression of XBP1 m RNA in CCK-8+LPS group was significantly decreased(P<0.05), and pretreatment with CCK-8-1 receptor antagonist devazepide can inhibit the effect of CCK-8 significantly. Compared with the control group, no significant difference in CCK-8 alone group or devazepide alone group was found in XBP1 m RNA expression levels(P>0.05). The results indicate that CCK-8 inhibited activation of LPS-induced RAW264.7 cell IRE1 by CCK-8 receptor 1. 3 The effect of CCK-8 on protein expression of GRP78 and CHOP in LPS-induced RAW264.7 cells GRP78 and CHOP are Protein biomarkers in ERS activation. Compared with the control group, the expressions of GRP78 and CHOP in LPS group were significantly increased(P<0.05). pretreatment with CCK-8 significantly inhibited the changes induced by LPS, which was attenuated by CCK-8-1receptor blocker devazepide. Compared with the control group, no significant difference in CCK-8 alone group or devazepide alone group was found in the expressions of GRP78 and CHOP(P>0.05). The results indicate that CCK-8 inhibited activation of LPS-induced RAW264.7 cell ERS by receptor 1. 4 CCK-8 inhibited release of IL-1βin LPS-induced RAW264. 7 cells Increased expression of CHOP in macrophages can cause increased release of IL-1β. Compared with the control group, expression of IL-1β in LPS group was significantly increased(P<0.05), pretreatment of CCK-8 could inhibit increased LPS-induced IL-1β levels. The pre-application of devazepide reversed the effect of CCK-8(P<0.05). Changes in IL-1β levels were consistent with CHOP protein expression levels.Conclusion:CCK-8 could inhibit ERS and the generation of IL-1βin LPS-induced RAW264.7 cell by its receptor 1, which might be the upstream mechanism of the inhibitory effect of CCK-8 on LPS-induced IL-1β expressions.