Discussion
In this study, we demonstrated for the first time that SBI could alleviate lupus-like manifestations and improve the efficacy of MSCs therapy in R848-induced mice. SBI synergized with MSCs to restore immune balance, particularly by suppressing aberrant Tfh cell production. On the molecular level, SBI increased the ability of MSCs to down-regulate the expression of Tfh differentiation-related genes, mainly through the inhibition of IL-6 and its downstream pathways.
So far, there is no study reported of SBI regulating Tfh cells. As the main active ingredient of silymarin, SBI has been previously reported to have immunomodulatory effects on Th1, Th17 cells[11[] Ribeiro, Vanessa Rocha et al. “Silibinin downregulates the expression of the Th1 and Th17 profiles by modulation of STATs and transcription factors in pregnant women with preeclampsia.” International immunopharmacology vol. 109 (2022): 108807. doi:10.1016/j.intimp.2022.108807]. It also increases the function of regulatory T cells through the immunomodulatory effect of up-regulation of FOXP3 expression, and can be used as an adjuvant therapy to alleviate the adverse effects associated with interferon-beta treatment in patients with multiple sclerosis (MS)[22[] Abbasirad, Faezeh et al. “Significant immunomodulatory and hepatoprotective impacts of Silymarin in MS patients: A double-blind placebo-controlled clinicaltrial.” International immunopharmacology vol. 97 (2021): 107715. doi:10.1016/j.intimp.2021.107715]. Ourin vivo and in vitrostudy suggests that SBI mainly regulates Tfh and Treg cells in lupus mice. Among them, the inhibition of Tfh cell production was largely achieved by suppressing its differentiation-related genes, while the effect on cell proliferation and apoptosis was not so evident. Overall, SBI is a promising immunomodulatory agent with a wide range of immunomodulatory functions under different conditions.
IL-6/STAT3 axis is critical in the development of Tfh cells[33[] Wan, Siyuan et al. “Costimulation molecules differentially regulate the ERK-Zfp831 axis to shape T follicular helper cell differentiation.” Immunity vol. 54,12 (2021): 2740-2755.e6. doi:10.1016/j.immuni.2021.09.018]. Consistent with literature reports[44[] Zheng, Rongjuan et al. “Chemopreventive Effects of Silibinin on Colitis-Associated Tumorigenesis by Inhibiting IL-6/STAT3 Signaling Pathway.” Mediators of inflammation vol. 2018 1562010. 25 Oct. 2018, doi:10.1155/2018/1562010], we experimentally verified the inhibitory effect of SBI on the IL-6/STAT3 pathway. Besides STAT3, IL-6 may also activate the downstream PI3K/mTOR pathway through the action of signal transducer glycoprotein (gp130)[55[] Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J. 2003 Aug 15;374(Pt 1):1-20. doi: 10.1042/BJ20030407]. The role of SBI on the mTOR pathway remains controversial, as a study examining the protective effects of SBI against cerebral ischemia showed that SBI treatment activated Akt/mTOR signaling[66[] Wang, Chaohui et al. “Protection by silibinin against experimental ischemic stroke: up-regulated pAkt, pmTOR, HIF-1α and Bcl-2, down-regulated Bax, NF-κB expression.” Neuroscience letters vol. 529,1 (2012): 45-50. doi:10.1016/j.neulet.2012.08.078], and another research on LPS stimulated porcine mammary epithelial cells showed SBI increased the expression of mTOR and S6[77[] Xu, Shengyu et al. “Silibinin Alleviates Lipopolysaccharide Induced Inflammation in Porcine Mammary Epithelial Cells via mTOR/NF-κB Signaling Pathway.” Molecular nutrition & food research vol. 67,14 (2023): e2200715. doi:10.1002/mnfr.202200715]. Our data indicate that SBI inhibits both STAT3 and mTOR pathways downstream of IL-6, thus further clarifying the molecular mechanism of SBI treatment.
A series of studies have shown a reduction in the dose of immunosuppressive drugs and a significant decrease in mortality in patients with SLE after MSC treatment[8][88[] Wang, Dandan et al. “Long-term safety of umbilical cord mesenchymal stem cells transplantation for systemic lupus erythematosus: a 6-year follow-up study.” Clinical and experimental medicine vol. 17,3 (2017): 333-340. doi:10.1007/s10238-016-0427-0], yet there are still many challenges to overcome before clinical application. MSCs have strong immunomodulatory plasticity and are susceptible to microenvironmental influences. Besides, MSCs can secrete cytokines with strong pro-inflammatory effects, such as IL-6, which may reduce the efficacy of MSC therapy in SLE patients[99[] Li, Aifen et al. “Mesenchymal Stem Cell Therapy: Hope for Patients With Systemic Lupus Erythematosus.” Frontiers in immunology vol. 12 728190. 30 Sep. 2021, doi:10.3389/fimmu.2021.728190]. In this study, we revealed that SBI synergized with MSCs to inhibit Tfh cell production. SBI could counteract IL-6 and its downstream pathways, thus may enhance the therapeutic effect of MSCs in lupus mice.
In summary, our data suggest that SBI is an effective and promising agent for the treatment of lupus autoimmunity. Moreover, SBI could improve the efficacy of MSCs in R848-induced lupus mice, which may provide a theoretical basis for better clinical application of MSCs. However, the prospective use of SBI in SLE patients still needs to be validated by further experimental and clinical evidence.