Novel Mechanisms of Herbal Therapies for Inhibiting HMGB1 Secretion or Action

High mobility group box 1 (HMGB1) is an evolutionarily conserved protein and is constitutively expressed in virtually all types of cells. In response to microbial infections, HMGB1 is secreted from activated immune cells to orchestrate rigorous inflammatory responses. Here we review the distinct mechanisms by which several herbal components inhibit HMGB1 action or secretion, such as by modulating inflammasome activation, autophagic degradation, or endocytic uptake. In light of the reciprocal interactions between these cellular processes, it is possible to develop more effective combinational herbal therapies for the clinical management of inflammatory diseases.


Introduction
High mobility group box 1 (HMGB1), an evolutionarily conserved 30 kDa DNA-binding protein, is ubiquitously expressed in virtually all types of cells. Bearing two nuclearlocalization sequences (NLS), HMGB1 is transported into the nucleus by the nuclear import complexes, thereby maintaining a large nuclear "pool" of preformed protein [1]. It carries two internal repeats of positively charged domains ("HMG boxes" known as "A box" and "B box") in the N-terminus and a continuous stretch of negatively charged (aspartic and glutamic acid) residues in the C-terminus. These HMG boxes enable HMGB1 to bind chromosomal DNA and fulfill its nuclear functions in stabilizing nucleosomal structure and regulating gene expression [1]. The disruption of local expression of HMGB1 renders animals susceptible to infectious [2] or injurious insults [3,4], reinforcing a beneficial role of intracellular HMGB1 in immunity against infection and injury [5].
In response to infections and injuries, however, HMGB1 is secreted from activated immune cells or passively released from injured cells. Excessive HMGB1 secretion/release adversely contributes to the pathogenesis of infectionand injury-elicited inflammatory diseases. For instance, in animal models of endotoxemia or sepsis (induced by cecal ligation and puncture, CLP), HMGB1-neutralizing antibodies improve survival [6] and rescue rodents from lethal sepsis even if given at 24 h after CLP [7,8]. Similarly, HMGB1-specific antibodies are protective against ischemia/reperfusion [9][10][11], trauma [12,13], chemical toxemia [14][15][16], atherosclerosis [17], gastric ulcer [18], and hyperoxia [19], supporting the pathogenic role of HMGB1 in injury-elicited inflammatory diseases. Furthermore, in animal models of rheumatoid arthritis, anti-HMGB1 agents confer significant protection against joint tissue edema [20][21][22], supporting a pathogenic role for HMGB1 in autoimmune diseases. The establishment of HMGB1 as a mediator of various inflammatory diseases has prompted the search for inhibitors that can attenuate HMGB1 secretion or action. In this review, we summarize the divergent mechanisms by which several herbal therapies effectively inhibit active HMGB1 secretion and action and hope to stimulate interests 2 Evidence-Based Complementary and Alternative Medicine in developing novel HMGB1-targeting therapeutic strategies for the treatment of inflammatory diseases.

Extracellular Role of HMGB1 as an Alarmin
Once released, extracellular HMGB1 functions as an alarmin signal to alert, recruit, and activate immune cells. For instance, HMGB1 binds to various microbial products (e.g., CpG-DNA or LPS), thereby facilitating their recognition by respective receptors to augment inflammatory responses [33].
As an evolutionarily conserved cellular process for degrading damaged cytoplasmic macromolecules, autophagy begins with the formation of double-membraned structures, which elongate and engulf portions of the cytoplasm to form autophagosomes. Subsequently, autophagosomes fuse with lysosomes to form degradative autophagolysosomes, where the engulfed contents are degraded by acidic lysosomal hydrolases. Indeed, EGCG can be trafficked into autophagosomes within 6 h and then destined to the lysosomal-associated membrane protein 2-(LAMP2-) containing autophagolysosomes within 16 h [98]. Meanwhile, EGCG conjugates to cytoplasmic HMGB1, leading to the formation of EGCG-HMGB1 complexes (dimmer, trimmer, tetramer, and oligomer) (Figure 4) [98]. This is consistent with previous findings that EGCG may conjugate to proteins either covalently with the free thiol group of cysteine residues [99] or noncovalently via hydrogen bonding, aromatic stacking, or hydrophobic interactions [100]. Because these large EGCG-HMGB1 complexes cannot physically pass through the narrow pore of the proteasome barrel of the ubiquitinproteasome pathway, they trigger the autophagic degradation process. Consistently, at the concentrations effective for inhibiting HMGB1 secretion, EGCG dramatically enhances the formation of autophagosomes [98]. In contrast, the coaddition of autophagy inhibitors (e.g., 3-methyladenine) impairs EGCG-mediated inhibition of HMGB1 secretion, thereby leading to a dramatic accumulation of HMGB1 aggregates in macrophages. Recently, EGCG has also been proven effective in stimulating autophagy in other cell types including breast cancer cells [98], hepatocytes [101], retinal pigment epithelial cells [102], and vascular endothelial cells [103]. Given the possibility that HMGB1 interacts with autophagy regulators (e.g., beclin-1) in the cytoplasm [104,105], it will be important to investigate whether HMGB1 occupies a critical role in EGCG-mediated autophagy. This is relevant because recent studies indicate that bacterial endotoxin induces significantly less autophagy in HMGB1deficient macrophages [2].

Tanshinone IIA Sodium Sulfonate (TSN-SS) Stimulates
Endocytic HMGB1 Uptake. Danshen is a medicinal herb that contains several red pigments including tanshinones I, II, and IV and cryptotanshinone, which exhibit various anti-inflammatory properties. Accounting for 5-6% of the total dry weight of Danshen root, tanshinone IIA dosedependently attenuates LPS-induced HMGB1 secretion, with an estimated IC 50 < 25 M. However, its poor water solubility may adversely affect the bioavailability and therapeutic efficacy of tanshinone IIA [55], thereby prompting the exploration of water-soluble derivatives as more effective HMGB1 inhibitors. One such compound, tanshinone IIA sodium sulfonate (TSN-SS), dose-dependently inhibits LPSinduced HMGB1 secretion with an estimated IC 50 < 10 M. At the doses that completely prevent HMGB1 secretion, TSN-SS does not affect endotoxin-induced release of most other cytokines and chemokines (such as IL-6, IL-12p40/p70, KC, MCP-1, MIP-1 , MIP-2, and TNF), indicating a selectivity for TSN-SS in inhibiting HMGB1 secretion.
Given the regulatory role of HMGB1 in autophagy [2,104,105], the TSN-SS-mediated HMGB1 endocytosis may be linked to autophagy ( Figure 5). When occurring simultaneously, endocytosis and autophagy can converge on a common lysosome-dependent pathway, leading to eventual HMGB1 degradation. Specifically, endosomes fuse with autophagosomes to form amphisomes [107,108], which then merge with lysosomes to form autolysosomes, where the amphisome contents are digested by lysosomal enzymes [109]. In the presence of TSN-SS, exogenous HMGB1 was detected in increased number of larger cytoplasmic vesicles that colocalized with autophagy-(LC3-positive) punctate structures, suggesting that HMGB1-containing endosomes may have been fused with autophagosomes to form amphisomes. The internalized HMGB1 is then possibly degraded via the lysosome-dependent pathway, because bafilomycin A1, a specific inhibitor of autophagosome-lysosome fusion, prevents the degradation of LC3-II and exogenous HMGB1. Taken together, these results suggest that TSN-SS facilitates endocytosis of exogenous HMGB1, leading to subsequent HMGB1 degradation via a lysosome-dependent pathway ( Figure 5). Notably, even when given several hours after the endotoxin stimulation, TSN-SS still effectively blocks HMGB1 secretion. It is thus possible to strategically administer TSN-SS in a delayed fashion to pharmacologically "recycle" injurious proinflammatory mediators (such as HMGB1) back to innate immune cells. TSN-SS has already been used in China as a medicine for patients with cardiovascular disorders, and its capacity to facilitate endocytic HMGB1 uptake by professional phagocytes may provide basis for the treatment of both infection-and injury-elicited inflammatory diseases [26]. : TSN-SS stimulates endocytic HMGB1 uptake. TSN-SS facilitated internalization of exogenous HMGB1 possibly via clathrin-and caveolin-dependent endocytosis into cytoplasmic vesicles that eventually mature into endosomes. Consequently, it likely triggers another cellular degradation process, autophagy, during which cytoplasmic macromolecules are engulfed by doublemembraned cytoplasmic vesicles termed autophagosomes. Subsequently, these HMGB1-containing endosomes could be fused with other cytoplasmic vesicles (such as autophagosomes) to form amphisomes, where the internalized HMGB1 was likely degraded via a lysosome-dependent mechanism.

Therapeutic Efficacy of HMGB1-Inhibiting Herbal Components
Given the capacity of various herbal components in preventing endotoxin-induced HMGB1 secretion, we explored their efficacy in animal models of CLP-induced sepsis.
Recently, a herbal remedy consisting of five herbs (Danggui, Danshen, Honghua, Chuanxiong, and Chishao) has been developed in China for treating septic patients. This combinational therapy, termed "Xuebijing, " has been proven to be protective experimentally in animal model of sepsis [126] or clinically in patients with sepsis [127,128]. In light of the distinct but occasionally complementary mechanisms of herbal inhibition of HMGB1 release or action, combinational therapy with multiple herbs might result in an improved therapeutic efficacy. For instance, the induction of autophagy by EGCG may provide a negative feedback regulation of inflammasome activation at multiple levels such as by eliminating damaged mitochondria (to prevent mitochondrial DNA release) [129], removing active inflammasomes [129,130], and destroying cytoplasmic HMGB1 [98]. It is thus important to test whether a better protection could be achieved by combinational therapy with several HMGB1 inhibitors that divergently modulate autophagy (e.g., EGCG) and inflammasome (e.g., CBX). These important studies may pave the road for future clinical studies to explore the therapeutic potential of additional herbal cocktail for the treatment of sepsis and other inflammatory diseases.

Conclusions
HMGB1 is secreted by activated macrophages/monocytes through complex mechanisms including PKR-dependent inflammasome activation and pyroptosis. A growing number of herbal components have been proven to be effective in inhibiting endotoxin-induced HMGB1 secretion through divergently distinct mechanisms such as preventing PKR/inflammasome activation, stimulating HMGB1 autophagic degradation, and enhancing endocytic HMGB1 uptake. In light of the intricate relationship between endocytosis, autophagy, and inflammasome activation, it is important to test whether a better protection could be achieved by combinational therapy with several anti-HMGB1 agents.

Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors' Contribution
Andrew H. Wu and Li He equally contributed to this paper.