It has been reported that HMGB1 can be secreted actively by macrophages induced by various PAMP, including the LPS . The active secretion of HMGB1 into the extracellular compartment during inflammation involves two essential steps. The first step involves the hyperacetylation of the lysine residues at the nuclear localizing sequence (NLS) of HMGB1 via the JAK-STAT signaling pathway, which promotes the shuttling of HMGB1 protein out from the nucleus into the cytoplasm . Then, the second step involves the secretion of the cytoplasmic HMGB1 into the extracellular compartment either via the pyroptosis or exocytosis of secretory lysosomes . Our current findings demonstrated that pretreatment with ZnC was able to inhibit the secretion of HMGB1 into the extracellular compartments. However, the exact mechanisms that underlying such observation remain unclear. Hence, future study is needed to determine how ZnC inhibit the secretion of HMGB1 during inflammation.
During inflammation, reactive oxygen species (ROS) are generated by the immune cells, leading to oxidative burst . The ROS produced may alter the functionality and biological activities of various proteins and enzymes, including the HMGB1. It has been reported that the pro-inflammatory properties of HMGB1 are greatly depending on the redox state of two redox-sensitive cysteine moieties (C23 and C45) . When these two cysteine moieties are in reduced condition, HMGB1 is immunologically inactive. However, excessive production of ROS during inflammation can oxidize the C23 and C45 residues to form a disulfide bond. The resulting conformation changes enable HMGB1 to bind to the TLR and act as a pro-inflammatory mediator. It has been reported that the GSH and thioredoxin can help to maintain HMGB1 in its reduced form after LPS induction in human umbilical vein endothelial cells (HUVEC) and HK-2 human kidney proximal tubule cells, hence limiting the pro-inflammatory activities of HMGB1 . Since GSH and thioredoxin are part of the non-enzymatic antioxidant defense mechanisms in the cells, these thiol-based antioxidants can prevent the HMGB1 protein from being oxidized by scavenging the ROS that being produced during inflammation . Our current results showed that ZnC was able to increase the free thiol level of RAW 264.7 cells after LPS induction, indicating that ZnC may help to limit the pro-inflammatory activities of HMGB1 by keeping it in the reduced state.
It has been reported that the binding of HMGB1 to the TLR can cause the activation of NF-κB signaling . NF-κB is a transcription factor that involves in the regulation of immune response, inflammation, and cell survival . The activation of the NF-κB can induce the expression of various inflammatory mediators, such as the tumor necrosis factor-alpha (TNF-α), interleukin-1, and interleukin-6. Hence, the HMGB1 protein released into the extracellular compartments may cause overproduction of such mediators. Besides, we demonstrated that pretreatment with ZnC was able to attenuate the late phase NF-κB activation, but not the early activation of NF-κB. Similar findings were reported in previous studies as well [13, 21]. This is reasonable since HMGB1 is a late mediator of sepsis pathogenesis due to its delayed released manner . Previously, it has been reported that the secretion of HMGB1 can only be detected in mice after 8 hours of LPS administration . Hence, it is possible that the suppression of NF-κB activation after 22 hours of LPS induction was partly due to the inhibition of HMGB1 secretion by ZnC pretreatment.
Interestingly, our current findings demonstrated that after 30 minutes of LPS induction, pretreatment with ZnC can enhance the early phase NF-κB activation instead of suppressing its activation. Following LPS stimulation, the TLR4 signals to the activation of the NF-κB via the MyD88 dependent or TRIF dependent signaling pathway . The MyD88 dependent pathway leads to the early phase NF-κB activation while the TRIF dependent pathway signals to the late phase NF-κB activation. Previously, it has been demonstrated that zinc, which is one of the subcomponents of ZnC, can regulate these two pathways differentially . Intracellular free zinc ions were demonstrated to enhance MyD88 dependent NF-κB activation while suppressing the TRIF dependent NF-κB activation in cells. Results from this present study are in agreement with previous findings where ZnC was found to enhance the early phase NF-κB activation while suppressing the late phase NF-κB activation. It is noted that the activation of NF-κB not only can induce the expression of pro-inflammatory mediators, but it may also induce the expression of anti-inflammatory mediators, including the A20 protein . A20 protein is a deubiquitinase that involved in the termination of the NF-κB signaling pathway . Previous study has demonstrated that zinc can downregulate the NF-κB signaling pathway and inhibit the expression of pro-inflammatory mediators via the induction of A20 protein . Hence, further study is needed to determine whether the induction of early NF-κB activation by ZnC pretreatment can lead to the expression of A20 protein and other anti-inflammatory mediators or not.
Furthermore, pretreatment with ZnC was showed to activate the Akt signaling pathway after 30 minutes of LPS induction. Previously, the activation of the Akt signaling pathway was demonstrated to limit the pro-inflammatory responses in LPS-induced macrophages by downregulating the TLR signaling cascade [28, 29]. Besides, the inhibition of activation of the Akt signaling pathway by using inhibitors greatly enhanced the cytokine production and reduced the survival time in LPS-induced endotoxemic mice . The Akt signaling pathway was also reported to be crucial in the polarization of macrophages toward the M2 phenotype . Hence, ZnC may exert its anti-inflammatory effects by activating the Akt signaling pathway.
In conclusion, pretreatment with ZnC can inhibit the secretion of HMGB1 protein from RAW 264.7 cells and suppress the activation of the late phase NF-κB signaling pathway after induction with LPS. Moreover, present findings also demonstrated that ZnC was able to enhance the activation of the Akt signaling pathway, suggesting that Akt may play a crucial role underlying the anti-inflammatory effects of ZnC. Taken together, we propose that ZnC is capable to prevent the occurrence of sepsis by inhibiting the secretion of HMGB1 from immune cells.