Bioactive compounds from ShenFuShanYuRou decoction enhance Treg cell function against hemorrhagic shock injury via Stat1‐ and Gbp5‐dependent FOXP3 induction

Dear Editor, In this study, we unveiled the bioactive compounds and molecular mechanisms of ShenFuShanYuRou decoction (SFSY) against hemorrhagic shock/resuscitation (HS/R) injury via the promotion of regulatory T (Treg) cell function. Our work offers new therapeutic strategies for circumventing HS/R-induced injury. HS is a substantial global problem with more than 1.9 million deaths per year worldwide.1 While advances in resuscitation strategies have circumvented early mortality from HS, still ∼30% of patients experience multiple organ dysfunction (MOD).2 Treg cells play a vital role in maintaining innate immune homeostasis to foster tissue repair.3 Thus, multitarget regulation of Treg cell function offers a new therapeutic intervention to minimize HS/R injury. SFSY is a famous Traditional Chinese Medicine formula, widely used in the supplementary therapy of patients with shock in China. However, the bioactive compounds and molecular mechanisms of SFSY against HS/R injury have not yet been elucidated. A total of 263 chemical compounds and 39 prototype compounds in the plasma of SFSY were characterized (Figure S1–S3, Tables S1–S3). To clarify the effect of SFSY treatment on Treg cell function, the function and frequency of Th, Ts, Th1, Th2, Th17, and Treg cells were detected in the well-established rodent model of HS/R (Figure S4A) and a naïve Treg cell model. As shown in Figure 1A, SFSY treatment did not affect the transcripts of Tbx21, Gata3, and Rorc, but increased FOXP3 transcript in response to HS/R. We also found that SFSY increased the proportion of Treg cells in both peripheral blood mononuclear cells (PBMCs) and lungs (Figure 1B,C; Figures S4B and S5). Furthermore, the incubation with SFSY increased the localization of FOXP3 to the nuclei of Treg cells (Figure 1D; Figure S6). These results indicate that SFSY treatment augments FOXP3 expression, thereby promoting Treg cell function both in in vitro and in vivo. Additionally, SFSY treatment increased the blood pressure and heart rate (Figure 2A,B; Figure S7A). The HS/R-induced metabolic disorders, lymphocyte depletion, and MOD (lungs, liver, kidneys, and intestine) injury were also ameliorated by SFSY treatment (Figure 2C–F; Figures S7B–S11). To explore the molecular mechanisms underlying the SFSY-mediated Treg cell function, CD4+CD25+ Treg cells were purified from PBMCs, and transcriptomic sequencing was performed. The results of principal component and volcano map analyses showed significant differences in mRNA expression among the Sham, HS/R, and HS/R + SFSY groups (Figure 2G,H; Figure S12). SFSY significantly reduced the HS/R-induced activation of immune-related pathways in Treg cells (Figure 2I; Figure S13A). Further validation studies in Treg cells and lungs demonstrated that the enhancement of Treg cell function by SFSY against HS/R-induced injury was Stat1-, Ebi3-, CXCL10-, and Gbp5-dependent manner (Figure 2J; Figures S13B–S15). Next, we screened the bioactive ingredients in SFSY by integrative pharmacological screen strategy based on ingredients in plasma and phenotype experiments. Ginsenoside Ro, hypaconitine, loganic acid, secologanin, and wogonin significantly decreased the mRNA levels of IL-6, TNF-α, and IL-1β in A549 cells under LPS incubation (Figure S16). Importantly, ginsenoside Ro decreased the expression of Stat1, hypaconitine reduced the levels of Stat1 and CXCL10, and loganic acid decreased the expression of Gbp5 both in A549 and Treg cells (Figure S17A). We also found that the addition of ginsenoside Ro, hypaconitine, or loganic acid increased the frequency of FOXP3+ cells as well as their surface expression of CD4 in Treg cells (Figure S17B,C). Stat1 can be translocated to both the nucleus and mitochondria after phosphorylation to regulate FOXP3 transcription and the mitochondria function of Treg cells.4, 5 Ginsenoside Ro or hypaconitine treatment inhibited the phosphorylation of Stat1 and promoted the expression of FOXP3 in naïve Treg cells, which were significantly blocked by fludarabine (Stat1 inhibitor; Figure 3A–C; Figure S18A). Additionally, incubation with fludarabine abolished the reduction of CXCL10 mRNA under hypaconitine incubation in Treg cells, indicating that hypaconitine inhibits the phosphorylation of Stat1 to reduce CXCL10 transcription and promote FOXP3 expression (Figure 3D). Considering that ginsenoside Ro did not affect CXCL10 transcription, we speculated that it could affect the mitochondrial function of Treg cells. We analyzed mitochondrial dynamic, mitophagy, mitochondrial biogenesis, mitochondrial apoptosis, and mitochondrial oxidative phosphorylation (OXPHOS) function, and found that ginsenoside Ro mainly augmented the mitochondrial biogenesis, balanced mitochondrial dynamic, and enhanced mitochondrial OXPHOS function in Treg cells in a Stat1-dependent manner (Figure 3E–H; Figure S18B,C). Taken together, these results suggest that both ginsenoside Ro and hypaconitine may inhibit the phosphorylation of Stat1, but their mechanisms of enhancing Treg cell function are different. Gbp5, a unique regulator of NLRP3 inflammasome activation in innate immunity, can promote GSDMD-mediated pyroptosis.6 Although loganic acid had an inhibitory effect on Gbp5 transcription (Figure S17A), our western blot experiment did not show a decrease in Gbp5 expression. Therefore, we used LPS to induce a pyroptosis condition to enhance the abundance of changes in Gbp5 protein. Indeed, we found increased Gbp5 expression and cleaved-GSDMD/GSDMD ratio in LPS-treated Treg cells, which was abolished by treatment with loganic acid (Figure 4A). Furthermore, Gbp5 knockdown significantly ablated the effects of loganic acid treatment on decreasing pyroptosis and enhancing Treg cell function (Figure 4B–D; Figure S19A). The calcein/PI dye staining and apoptosis analysis confirmed that the circumvention of Gbp5-mediated pyroptosis was the substantial mechanism of loganic acid treatment on Treg cell survival (Figure 4E,F; Figure S19B). In conclusion, our study demonstrated that SFSY treatment promoted the stability of FOXP3, thereby enhancing Treg cell function and alleviating HS/R-induced metabolic disorders, lymphopenia, and MOD. Mechanistically, we revealed the following: (1) ginsenoside Ro circumvented the translocation of phosphorylated Stat1 to mitochondria, thereby increasing the mitochondrial function of Treg cells; (2) hypaconitine inhibited the phosphorylation of Stat1, thereby reducing CXCL10 transcription and promoting FOXP3 expression; and (3) loganic acid mitigated the activation of Gbp5 to inhibit Treg cell pyroptosis mediated by GSDMD cleavage (Graphical abstract). Our research illustrates that bioactive compounds from SFSY enhance Treg cell function against HS/R injury via Stat1- and Gbp5-dependent FOXP3 induction. Qingxia Huang: Investigation, data curation, methodology, validation, writing-original draft, funding acquisition. Mingxia Wu: Data curation, investigation, formal analysis, writing-original draft, visualization. Lu Ding: Investigation, formal analysis, validation, methodology. Chen Guo: Resources, data analysis, validation. Yisa Wang: data analysis, validation. Zhuo Man: Software, investigation. Hang Su: Data analysis, visualization. Jing Li: Investigation, validation. Jinjin Chen: Investigation, methodology. Yao Yao: Methodology. Zeyu Wang: Project administration. Daqing Zhao: Writing-review & editing. Linhua Zhao: Supervision, methodology, data analysis, writing-review & editing. Xiaolin Tong: Methodology, writing-review & editing, supervision, funding acquisition. Xiangyan Li: Conceptualization, supervision, methodology, resources, funding acquisition, writing-review & editing. This work was supported by the National Natural Science Foundation of China (grant no. 82374078 and 82104432), the Health Technology Capability Enhancement Project of Jilin Province (grant no. 2022JC041), and the Youth Excellent Discipline Talent Training Project (grant no. 202331), and the Innovation and Entrepreneurship Talent Funding Project of Jilin Province (grant no. 2022ZY10). The authors declare no conflict of interest. Animal protocols were approved by the Animal Ethics Committee of Changchun University of Chinese Medicine (Changchun, China, approval no. 2022433). All animal experiments used in this study were performed strictly in accordance with the ARRIVE guidelines 2.0 and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The data supporting the findings of this study are available from the corresponding authors upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.