Objective To evaluate mitochondrial mechanism of heme oxygenase-1(HO-1)in hibiting the synthesis of tumor necrosis factor-α(TNF-α), interleukin-1β (IL-1β) and reactive oxygen species (ROS) in RAW264.7 macrophages.
Methods RAW264.7 macrophages were cultured in vitro and randomly divided into six groups including blank control group (group C), lipopolysaccharide (LPS) model group (CL group), hemin (HO-1 inducer) +LPS group (HL group), zinc proporphyrin-IX (ZnPP-IX, HO-1 antagonist) +LPS group (ZL group), 3-methyladenine (3-MA, mitochondrial autophagy inhibitor) +LPS group (ML group), rapamycin (RAPA, mitochondrial autophagy promoter) +LPS group (RL group). Western blot was used to detect the relative expression levels of HO-1, TNF-α, IL-1β, microtubule-associated protein 1A/1B light chain 3B (LC3B), mitochondrial fission protein 1 (FIS1) and relative quantity dynamin-related protein 1 (DRP1), and ROS assay kit was used to detect the ROS content.
Results Compared with the group C, the expression levels of HO-1, LC3B and ROS in the other five groups were increased, and the expression level of DRP1 decreased (P < 0.05). Compared with the group C, FIS1 expressions in the CL group, ZL group and ML group increased, while FIS1 expression in the HL group and RL group decreased, the differences were statistically significant (P < 0.05). Compared with the C group, the expression levels of IL-1β and TNF-α in the HL group were decreased, the differences were statistically significant (P < 0.05). Compared with the CL group, LC3B expression in the HL group was decreased, while LC3B expressions in the ZL group, ML group and RL group were increased, the differences were statistically significant (P < 0.05). Compared with the CL group, the expression levels of TNF-α and IL-1β in the HL group were decreased, the expression level of IL-1β in the RL group was increased and level of TNF-α was decreased, while the expression levels of TNF-α and IL-1β in the ML group were increased, the differences were statistically significant (P < 0.05). Compared with the CL group, ROS expression levels in the HL and RL groups were decreased, while were increased in the ZL and ML groups, the differences were statistically significant (P < 0.05). The expression levels of TNF-α, IL-1β and DRP1 in the HL group were lower than those in the other five groups, while the expression level of HO-1 was higher than that in the other five groups, the differences were statistically significant (P < 0.05). The expression level of FIS1 in the RL group was lower than that in the HL group, and the expression level of LC3B was higher than that in the HL group (P < 0.05). The expression levels of IL-1β, TNF-α, FIS1, DRP1 and ROS in the RL group were lower than those in the ML group, while the expression level of LC3B was higher than those in the ML group, the differences were statistically significant (P < 0.05).
Conclusion HO-1 can inhibit the mitochondrial division of RAW264.7 macrophages thereby reducing the expression of inflammatory factors, and the effect is better than that of mitochondrial autophagy.
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