SSA is a polysaccharide extracted from Radix bupleuri that regulates LXRα(2),and has a variety of pharmacological activities, especially anti-inflammatory and antitumor effects (18). LXRα is a subtype of LXR, that is mainly expressed in tissues related to lipid metabolism, such as liver, small intestine, spleen,and adipose tissue(19). It is also expressed in lymphocytes and macrophages, and is involved in inflammatory responses and the transcriptional activation of macrophages and MDSCs(20). Studies have shown that LXR agonists such as GW3965 and RGX-104 effectively induce the expression of the target genes ATP-binding cassette transporter A1A1(ABCA1) and apolipoprotein E (ApoE), thereby inhibiting the survival of some immune cells. Deletion of LXRα promoted the differentiation and survival of MDSCs, and exerted immunosuppressive effects to alleviate hepatic injury(12). The LXR agonist GW3965 induced MDSCs apoptosis by activating ApoE to inhibit the survival of MDSCs, and decrease the number of MDSCs in the TME, peripheral blood, and spleen in vitro and in vivo, thereby reducing the inhibitory function of MDSCs on T cells to exert antitumor effects(13). Therefore, LXR agonists, which regulate MDSCs, have emerged as novel tumorimmunotherapies.
Our results show that the level of LXRα in MDSCs significantly increased with SSA concentration (Fig. 1A-F). Similarly, the mRNA expression level of LXRα was also higer in MDSCs treated with 2.5 mg/L SSA than in those not treated with SSA (Figure). Our results confirmed that SSA can upregulte LXRα expression in MDSCs.
Studies have shown that deletion of LXRα promoted the differentiation and survival of MDSCs, that LXR agonists can induce apoptosis of MDSCs by activating LXR transcriptional targets, thereby inhibiting the immunosuppressive function of MDSCs(13). During the induction of MDSCs differentiation, our results indicated that the SSA can regulate the differentiation of MDSCs (Fig. 2A-G), and promote the apoptosis of MDSCs (Fig. 2H-K). Given our findings above that SSA can upregulate LXRα expression levels of MDSCs (Fig. 1). These results indicated that SSA can regulate the differentiation and apoptosis of MDSCs by upregulating LXRα expression.
Although both M-MDSCs and PMN-MDSCs have immunosuppressive functions, in terms of functional differences, M-MDSCs and PMN-MDSCs exert immunosuppressive effects through different mechanisms: M-MDSCs can upregulate the expression of ARG-1, iNOS and TGF-β mainly through the STAT1 pathway; however, PMN-MDSCs can upregulate the activation of NADPH and affect the expression of ROS, thus exerting immunosuppressive functions (21, 22). Both MDSCs types can exert inhibitory effects through ARG-1(23). L-arginine in the surrounding environment is depleted by the secretion of ARG-1, which not only limits the formation of cD3ξ chains on the TCR, but also inhibits the expression of cyclin D3 (CCND3) and cyclin-dependent kinase 4 (CDK4), thus, T-cell arrest in the G0-G1 phase of the cell cycle, ultimately inhibits proliferation of T cells (24). Certainly, both MDSCs can also suppress T cell immune responses by secreting ROS(25). The LXR agonists can inhibite the immunosuppressive function of MDSCs by activating LXR transcriptional targets (13). Our results showed that the levels of ROS and ARG-1(Fig. 3A-E, G, H) in MDSCs were significantly decreased in the SSA treated group, and the 2.5 mg/L SSA had the greatest effect. Similarly, the mRNA expression level of ARG-1 was also significantly decreased in MDSCs treated with 2.5 mg∕L SSA (Fig. 3F). These results showed that SSA can inhibit the immunosuppressive ability of MDSCs. This may be related to the upregulation of LXRα expression.
The administration of SSA by gavage and intraperitoneal injection is common, and most studies have investigated the effect of SSA on the treatment and improvement of this disease by establishing mouse or rat disease models. ZHAO et al(6),used in a model of 7,12-dimethyl-benz [a] anthracene (DMBA) induced breast cancer, and found that the administration of SSA via gavage decreased the level of the anti-inflammatorycytokine IL-10, while the anti-inflammatory and antifibrotic actions of SSA on CCl4-induced liver damage, increased the level of IL-10 after the intraperitoneal injection of SSA(26).
Studies have shown that SSA has a very poor absorption capacity in the gastrointestinal tract and low bioavailability when administered orally (17). Therefore, in vivo, we observed changes in the proportion of immune cells in mice treated with two administration modalities, namely, SSA gavage (ig) and SSA intraperitoneal injection (ip). In vivo, our results showed that the proportion of MDSCs were significantly decreased, in which the proportion of M-MDSCs were decreased significantly, but there was no change in PMN-MDSCs (Fig. 5), and together with the proportion of CD8 + T cells, Mo/Mø cells and B cells were significantly increased, while total T cells, γδT, NK cells and Treg cells did not change, and the proportion of CD4 + T cells decreased in the SSA ig group (Fig. 5–7). The proportion of splenic MDSCs were significantly decreased, in which the proportion of M-MDSCs were decreased significantly, but there was no change in PMN-MDSCs (Fig. 5), and together with total T cells were significantly increased, in which the proportion of CD4 + T cells did not change, while the proportion of CD8 + T cells increased, Mo/Mø were significantly increased; while B cells, γδT, NK cells and Treg cells did not change in the SSA ip group (Fig. 5–7).
Our results show that although both modes of administration have inhibitory effects on MDSCs, the regulatory effects on other immune cells are not entirely consistent. Studies have shown that SSA first undergoes a series of different degrees of transformation in the stomach and intestine after oral administration, for example, SSA can metabolized into saikosaponin b1 and saikosaponin g under acidic gastric conditions, and can also be decomposed into saikosapogenin f and saikosapogenin f by the gut microflora hydrolases Bifidobacterium Saiko-1, and − 2 and Eubacterium sp A-44, resulted in poor intestinal permeability and low bioavailability(27, 28). This may be the reason for the inconsistent results with intraperitoneal injection, and the specific mechanism involved needs to be further explored.
Studies have shown that the STAT1 signaling pathway is essential for the function of MDSCs, which can participate in the regulation of ARG-1 and iNOS and block IFN-γ secretion by T cells, inhibiting T-cell proliferation(29).The NF-κB signaling pathway is closely associated with LXR. A study of cerebral ischemia reperfusion revealed(16) that lysophosphatidic acid (LPA) can decrease the expression level of LXR, and promote the release of inflammatory factors via the NF-κB signaling pathway, aggravating the cerebral reperfusion injury. Our results showed that SSA can inhibit the phosphorylation of STAT1 and NF-κB p65 (Fig. 4). This suggests that SSA increase the LXRα expression, inhibits the immunosuppressive function of MDSCs, and induces the apoptosis of MDSCs, which may be associated with the STAT1 and NF-κB p65 signaling pathways.