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\begin{document}

\title{Silybin acts synergistically with mesenchymal stem cells to ameliorate
R848-induced lupus-like disease in mice}



\author[1]{Nan Zhao}%
\author[1]{Miheraiy Abdukiyum}%
\author[1]{Yuanyuan Zheng}%
\author[2]{wenjing Li}%
\author[1]{Mengxi Huang}%
\author[2]{Mei Li}%
\author[2]{Tohtihan Alim}%
\author[1]{Yiyuan Cui}%
\author[3]{Xie Ying}%
\author[2]{Xiaojun Tang}%
\author[1]{Xuebing Feng}%
\affil[1]{Department of Rheumatology and Immunology，Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine}%
\affil[2]{Department of Rheumatology and Immunology，Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School，Nanjing University}%
\affil[3]{The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objective:~To determine the therapeutic effects of silybin (SBI) and its
synergistic effects with mesenchymal stem cells (MSCs) in a lupus mouse
model and to explore the therapeutic mechanisms. Methods:~TLR7/8 agonist
resiquimod (R848) was applied for the induction of lupus mice.
R848-induced B6 mice were randomly divided into a normal saline control
group, SBI group, MSCs group, and SBI plus MSCs group, and treated with
daily~SBI by gavage or received MSCs injection once via the tail vein.
Mice were sacrificed~at week 12, with urine, serum,~kidney, and spleen
collected.~The proportion of cell subsets was detected by flow cytometry
using splenocytes. Results: SBI treatment significantly decreased total
IgG, anti-ds-DNA antibody, and urinary protein levels, as well as renal
IgG and C3 deposition in R848-induced mice. It also increased the
ability of MSCs to suppress splenomegaly and serum antinuclear antibody
levels.~In vivo~and~in vitro~studies showed a decrease in the percentage
of Tfh cells after SBI treatment, which was most pronounced when
combined with MSC therapy.~When splenocytes of R848-induced mice were
treated with SBI and MSCs~in vitro, the expression of genes related to
Tfh cell differentiation, including~IL-6, Stat3, and~Bcl-6, was reduced,
and the phosphorylation of AKT, S6, and STAT3 proteins in Tfh cells was
decreased. Conclusion:~SBI acts synergistically with MSCs to ameliorate
lupus-like features in R848-induced mice.~It may enhance the ability of
MSCs to inhibit Tfh cell production by counteracting the activation of
IL-6 and its downstream pathways.%
\end{abstract}%



\sloppy


\textbf{Silybin acts synergistically with mesenchymal stem cells to
ameliorate R848-induced lupus-like disease in mice}

\textbf{Nan Zhao\textsuperscript{1},Miheraiy
Abdukiyum\textsuperscript{1}, YuanyuanZheng\textsuperscript{1}, Wenjing
Li\textsuperscript{2}, Mengxi Huang\textsuperscript{1}, Mei
Li\textsuperscript{2}, Tohtihan Alim\textsuperscript{2}, Yiyuan
Cui\textsuperscript{1}, Ying Xie\textsuperscript{3},Xiaojun Tang}
\textsuperscript{\textbf{2*}}\textbf{and
XuebingFeng\textsuperscript{12*}}

\textsuperscript{\textbf{1}} Department of Rheumatology and Immunology,
Nanjing Drum Tower Hospital Clinical College, Nanjing University of
Chinese Medicine, Nanjing.

\textsuperscript{\textbf{2}} Department of Rheumatology and Immunology,
Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School,
Nanjing University

\textsuperscript{\textbf{3}} State Key Laboratory of Quality Research in
Chinese Medicines, Macau University of Science and Technology, Macau.

\textsuperscript{\textbf{*}}\textbf{Corresponding to}

Xuebing Feng, M.D. and Ph.D., Department of Rheumatology and Immunology,
the affiliated Nanjing Drum Tower Hospital of Nanjing University Medical
School, Nanjing 210008, P.R.China (email: fengxuebing@hotmail.com)

Xiaojun Tang, Ph.D., Department of Rheumatology and Immunology, Nanjing
Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing
University, Nanjing 210008, China (email: xjtang09@163.com)



\textbf{Highlights:}

\begin{itemize}
\tightlist
\item
  Silybin relieved lupus-like disease in TLR7/8 agonist (R848)-induced
  mice.
\item
  Silybin increased the efficacy of mesenchymal stem cells (MSCs) in the
  treatment of lupus mice.
\item
  Silybin synergized with MSCs to inhibit Tfh cell production, which was
  mostly achieved by suppressing the expression of Tfh differentiation
  genes such as IL-6 and STAT3.
\item
  The effects of silybin on Tfh cells could be counteracted by the
  activation of IL-6 and its downstream pathways.
\end{itemize}

\textbf{Abstract} \textbf{:}

\textbf{Objective:}To determine the therapeutic effects of silybin (SBI)
and its synergistic effects with mesenchymal stem cells (MSCs) in a
lupus mouse model and to explore the therapeutic mechanisms.

\textbf{Methods:} TLR7/8 agonist resiquimod (R848) was applied for the
induction of lupus mice. R848-induced B6 mice were randomly divided into
normal saline control group, SBI group, MSCs group and SBI plus MSCs
group, and treated with daily SBI by gavage or received MSCs injection
once via the tail vein. Mice were sacrificed at week 12, with urine,
serum, kidney and spleen collected. The proportion of cell subsets was
detected by flow cytometry using splenocytes. Quantitative PCR and
western blot were used to detect mRNA and protein levels. The regulatory
mechanism of SBI on follicular helper T (Tfh) cells was explored
through\emph{in vitro} splenocyte experiments.

\textbf{Results} : SBI treatment significantly decreased total IgG,
anti-dsDNA antibody and urinary protein levels, as well as renal IgG and
C3 deposition in R848-induced mice. It also increased the ability of
MSCs to suppress splenomegaly and serum antinuclear antibody levels.
\emph{In vivo} and \emph{in vitro}studies showed a decrease in the
percentage of Tfh cells and an increase in the percentage of regulatory
T (Treg) cells after SBI treatment, which was most pronounced when
combined with MSC therapy. SBI inhibited Tfh cell production in a
dose-dependent manner. When splenocytes of R848-induced mice were
treated with SBI and MSCs \emph{in vitro} , the expression of genes
related to Tfh cell differentiation, including\emph{Icos, Stat3} and
\emph{Bcl-6} , was reduced, and the phosphorylation of AKT, S6, and
STAT3 proteins in Tfh cells was decreased. Correspondingly, addition of
IL-6 or STAT3/mTOR agonists to the culture system completely or
partially blocked the inhibitory effect of SBI on Tfh cells.

\textbf{Conclusion:} SBI acts synergistically with MSCs to ameliorate
lupus-like features in R848-induced mice. It may enhance the ability of
MSCs to inhibit Tfh cell production by counteracting the activation of
IL-6 and its downstream pathways.

\textbf{Keywords:}

Systemic lupus erythematosus; Silybin; Mesenchymal stem cells;
Follicular helper T cells; Interleukin-6

\textbf{Introduction} :

Systemic lupus erythematosus (SLE) is a complex autoimmune disease
manifested by the damage of multiple organs and
systems\textsuperscript{{[}}11{[}{]} Relle, Manfred et al. ``Epigenetic
Aspects of Systemic Lupus Erythematosus.''~Rheumatology and therapy~vol.
2,1 (2015): 33-46. doi:10.1007/s40744-015-0014-y\textsuperscript{{]}}.
The pathogenesis of SLE is characterized by the abnormal activation of T
and B lymphocytes, and the formation of
autoantibodies\textsuperscript{{[}}22{[}{]} Lou, Hantao et al.
``Autoantibodies in systemic lupus erythematosus: From immunopathology
to therapeutic target.''~Journal of autoimmunity~vol. 132 (2022):
102861. doi:10.1016/j.jaut.2022.102861\textsuperscript{{]}}. Among
which, the imbalance between regulatory T (Treg) cells and follicular
helper T (Tfh) cells has attracted much
attention\textsuperscript{{[}}33{[}{]} He, Jing et al. ``Low-dose
interleukin-2 treatment selectively modulates CD4(+) T cell subsets in
patients with systemic lupus erythematosus.''~Nature medicine~vol. 22,9
(2016): 991-3. doi:10.1038/nm.4148\textsuperscript{{]}}. Tfh cells
represent a distinct subset of CD4+ T cells specialized in providing
help to B cells and are required for the formation of germinal centers.
In lupus, abnormal proliferation and function of Tfh cells will lead to
pathological processes like autoantibody production and tissue damage,
and thus become a promising therapeutic target
\textsuperscript{{[}}44{[}{]} Feng, Xuebing et al. ``Inhibition of
aberrant circulating Tfh cell proportions by corticosteroids in patients
with systemic lupus erythematosus.''~PloS one~vol. 7,12 (2012): e51982.
doi:10.1371/journal.pone.0051982\textsuperscript{{]}{[}}55{[}{]} Wei,
Xindi, and Xiaoyin Niu. ``T follicular helper cells in autoimmune
diseases.''~Journal of autoimmunity~vol. 134 (2023): 102976.
doi:10.1016/j.jaut.2022.102976\textsuperscript{{]}}.

Our previous studies have shown that MSCs could suppress Tfh cell
differentiation and proliferation, which was mediated by inducible
nitric oxide synthase (iNOS) \textsuperscript{{[}6{]}}. Recently, it has
been proven that bone marrow-derived mesenchymal stem cells (MSCs) from
SLE patients were inefficient to maintain Treg and Tfh balance, while
transplantation of healthy MSCs effectively inhibited the expansion of
Tfh cells and alleviate lupus symptoms\textsuperscript{{[}}66{[}{]}
Zhang, Zhuoya et al. ``Human Umbilical Cord Mesenchymal Stem Cells
Inhibit T Follicular Helper Cell Expansion Through the Activation of
iNOS in Lupus-Prone B6.MRL-Fas~Mice.''~Cell transplantation~vol. 26,6
(2017): 1031-1042.
doi:10.3727/096368917X694660\textsuperscript{{]}{[}}77{[}{]} Jang,
Eunkyeong et al. ``Infusion of Human Bone Marrow-Derived Mesenchymal
Stem Cells Alleviates Autoimmune Nephritis in a Lupus Model by
Suppressing Follicular Helper T-Cell Development.''~Cell
transplantation~vol. 25,1 (2016): 1-15.
doi:10.3727/096368915X688173\textsuperscript{{]}}. However, this
treatment is only effective for a subset of
patients\textsuperscript{{[}}88{[}{]} Wang, Dandan et al. ``A Long-Term
Follow-Up Study of Allogeneic Mesenchymal Stem/Stromal Cell
Transplantation in Patients with Drug-Resistant Systemic Lupus
Erythematosus.''~Stem cell reports~vol. 10,3 (2018): 933-941.
doi:10.1016/j.stemcr.2018.01.029\textsuperscript{{]}}, for reasons that
may be related to the insufficient inhibitory effect or short
persistence time\textsuperscript{{[}}99{[}{]} Nie M, Chen G, Zhao C, Gan
J, Alip M, Zhao Y, Sun L. Bio-inspired adhesive porous particles with
human MSCs encapsulation for systemic lupus erythematosus treatment.
Bioact Mater. 2020 Aug 10;6(1):84-90.~\textsuperscript{{]}}. Therefore,
it is necessary to find effective strategies to maximize the therapeutic
effect of MSCs.

Silybin (SBI), a flavonoid extracted from the seeds and fruits of
silymarin in the compositae\textsuperscript{{[}}1010{[}{]} K\selectlanguage{polish}ř\selectlanguage{english}en V,
Valentov\selectlanguage{ngerman}á K. Silybin and its congeners: from traditional medicine to
molecular effects. Nat Prod Rep. 2022 Jun 22;39(6):1264-1281. doi:
10.1039/d2np00013j.\textsuperscript{{]}}, is mainly used as a
liver-protective drug in clinical
practice\textsuperscript{{[}}1111{[}{]} ederico, Alessandro et al.
``Evaluation of the Effect Derived from Silybin with Vitamin D and
Vitamin E Administration on Clinical, Metabolic, Endothelial
Dysfunction, Oxidative Stress Parameters, and Serological Worsening
Markers in Nonalcoholic Fatty Liver Disease Patients.''~Oxidative
medicine and cellular longevity~vol. 2019 8742075. 15 Oct. 2019,
doi:10.1155/2019/8742075\textsuperscript{{]}}, while studies have shown
it has anti-tumor and anti-diabetic
effects\textsuperscript{{[}}1212{[}{]} Wei, Zibo et al. ``Silybin
suppresses ovarian cancer cell proliferation by inhibiting isocitrate
dehydrogenase 1 activity.''~Cancer science~vol. 113,9 (2022): 3032-3043.
doi:10.1111/cas.15470\textsuperscript{{]}{[}}1313{[}{]} Liu, Panwen et
al. ``Silibinin ameliorates STZ-induced impairment of memory and
learning by up- regulating insulin signaling pathway and attenuating
apoptosis.''~Physiology \& behavior~vol. 213 (2020): 112689.
doi:10.1016/j.physbeh.2019.112689\textsuperscript{{]}}. Recently,
evidence has suggested that SBI has a therapeutic effect on
collagen-induced arthritis models and patients with rheumatoid
arthritis\textsuperscript{{[}}1414{[}{]} Tong, W W et al. ``Silibinin
alleviates inflammation and induces apoptosis in human rheumatoid
arthritis fibroblast-like synoviocytes and has a therapeutic effect on
arthritis in rats.''~Scientific reports~vol. 8,1 3241. 19 Feb. 2018,
doi:10.1038/s41598-018-21674-6\textsuperscript{{]}{[}}1515{[}{]} Xie,
Ying et al. ``Suppression of up-regulated LXR\selectlanguage{greek}α \selectlanguage{english}by silybin ameliorates
experimental rheumatoid arthritis and abnormal lipid
metabolism.''~Phytomedicine : international journal of phytotherapy and
phytopharmacology~vol. 80 (2021): 153339.
doi:10.1016/j.phymed.2020.153339\textsuperscript{{]}}, which opens new
perspectives for the treatment of systemic autoimmune diseases.
Moreover, SBI could up-regulate the gene expression of MSC-related
markers in a pulmonary arterial hypertension rat
model\textsuperscript{{[}}1616{[}{]} Zhang, Tingting et al. ``Silibinin
Upregulates CXCR4 Expression in Cultured Bone Marrow Cells (BMCs)
Especially in Pulmonary Arterial Hypertension Rat Model.''~Cells~vol.
9,5 1276. 21 May. 2020, doi:10.3390/cells9051276\textsuperscript{{]}},
suggesting that it may also have an impact on the function of MSCs. In
this study, we systematically observed the efficacy of SBI alone and in
combination with MSCs in the treatment of lupus mice for the first time
and elaborated its regulatory mechanisms on Tfh cells.

\textbf{Materials and methods}

\textbf{Mice models}

Four-week-old female C57BL/6 mice (B6) were purchased from Beijing
Spefford Biotechnology Co. Ltd (China) and maintained in the specific
pathogen-free grade environmental animal facility of Nanjing Drum Tower
Hospital. All mice were adaptive fed for one week, and then randomly
divided into two groups: acetone control group and TLR7/8 agonist
resiquimod (R848)-induced group. To induce lupus-like disease, 40ul R848
(2 mg/ml), dissolved in acetone and stored a -80\selectlanguage{ngerman}°C, was applied to the
auricle of one side of mice every 3 days for 7 weeks. All animal
experiments were approved by the Medical Ethics Committee of Nanjing
Drum Tower Hospital.

\textbf{Study design}

The experimental grouping of mice was shown in Table S1. Thirty-six
R848-induced B6 mice were randomly divided into 4 groups (n= 9 in each
group): normal saline treatment group, SBI treatment group, MSCs
treatment group, and SBI plus MSCs treatment group. Umbilical cord
derived MSCs were kindly provided by the Stem Cell Bank of Jiangsu
Province (Beike Biotechnology) and cultured as previously described
{[}11{[}{]} Yuan, Xinran et al. ``Mesenchymal stem cell therapy induces
FLT3L and CD1c+~dendritic cells in systemic lupus erythematosus
patients.''~Nature communications~vol. 10,1 2498. 7 Jun. 2019,
doi:10.1038/s41467-019-10491-8{]}. Both SBI and MSCs were suspended in
normal saline. SBI was administered by gastric gavage at 200mg/kg daily
from week 5, and MSCs (1×10\textsuperscript{6} cells) were injected via
the caudal vein at week 9. Meanwhile, acetone-induced B6 mice (n=6) were
applied as normal controls and received normal saline treatment. All
mice were sacrificed by cervical dislocation at week 12. Prior to this
urine was collected by massaging the bladder for urinary protein and
creatinine, and later serum was collected for antibody levels and
kidneys were collected to assess histologic changes. In addition, mouse
spleens were ground to cell suspensions, resuspended in red blood cell
lysis buffer for 5 min, passed through a 70\selectlanguage{greek}μ\selectlanguage{english}m cell filter, and
centrifuged to obtain splenocytes.

\textbf{Flow cytometry}

For surface staining, mouse-derived splenocytes were labeled for 30 min
with directly labeled antibodies against cell surface antigens. For
intracellular cytokine staining, cells were stimulated with 0.1 mg/ml
foponol-1-myristate-37-acetate (PMA) plus 11 ng/ ml ionomycin for 4 h at
37\selectlanguage{ngerman}°C (all from Enzo Life Sciences, Farmingdale, NY, USA), followed by
fixation and permeabilization with BD Cytofix kits (BD Biosciences. the
USA) to fix and permeabilize the cells, followed by staining with
corresponding antibodies. To stain intranuclear factor Foxp3, membranes
were broken for 30 min using a fixation/permeability kit (Invitrogen,
USA) and then stained with APC-anti-Foxp3. To assess the cell viability,
cells were directly stained with the reactive dye eFluor 506 for 20 min.
For cell proliferation and apoptosis, intranuclear staining with the
proliferation marker Ki67 was performed for 30 minutes, and cell
apoptosis was assessed with Annexin V-PE/7-AAD Apoptosis Detection Kit
(Vazyme Biotech, Nanjing). All antibodies used for flow cytometry
detection were listed in Table S2. The assays were performed on a BD
FACSAria III flow cytometer (BD Biosciences), and the data were analyzed
using FlowJo X software.

\textbf{Laboratory test}

Proteinuria was quantitatively detected by a urine protein detection kit
(Nanjing Jiancheng, China). Serum and urine creatinine levels were
measured by a creatinine detection kit (Nanjing Jiancheng, China). Urea
nitrogen levels were measured using a urea nitrogen kit (Nanjing
Jiancheng, China). Immunoglobulin G (IgG) and antibody levels were
measured using mouse IgG (Multi Sciences, China) and anti-nuclear
antibody (ANA), anti-double strand DNA (dsDNA) (Aifang Bioscience,
China) ELISA kits.

\textbf{Renal histology and immunofluorescence}

Renal tissue samples fixed in formaldehyde fixative were embedded in
paraffin and cut into 10\selectlanguage{greek}μ\selectlanguage{english}m sections. Hematoxylin-eosin (H\&E) staining
was used to observe the changes in glomeruli under the microscope refer
to the previous description\textsuperscript{{[}}11{[}{]} Chen, Weiwei et
al. ``Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell
Differentiation.''~Journal of the American Society of Nephrology :
JASN~vol. 31,10 (2020): 2263-2277.
doi:10.1681/ASN.2019090937\textsuperscript{{]}}. Immunofluorescence
staining of paraffin-fixed kidney sections with Rhodamine conjugated
anti-IgG (Jackson Immuno Research, USA) and FITC conjugated
anti-complement 3 (C3) (Abcam, UK) antibodies was performed to observe
IgG and C3 deposition under a fluorescence microscope, as we described
previously\textsuperscript{{[}}22{[}{]} Zhang, Zhuoya et al.
``Mesenchymal stem cells prevent podocyte injury in lupus-prone
B6.MRL-Faslpr mice via polarizing macrophage into an anti-inflammatory
phenotype.''~Nephrology, dialysis, transplantation : official
publication of the European Dialysis and Transplant Association -
European Renal Association~vol. 34,4 (2019): 597-605.
doi:10.1093/ndt/gfy195\textsuperscript{{]}}.

\textbf{Polymerase chain reaction (PCR)}

RNA was extracted from mouse splenocytes by Trizol reagent (Vazyme
Biotech, Nanjing, China) and reverse transcribed by using Superscript
qRT SuperMix kit (Vazyme Biotech) according to the manufacturer's
instructions. All the primers were synthesized by GenScript (Nanjing,
China) and are listed in Table S3. Quantitative real-time RT-PCR
(qRT-PCR) was performed by using the SYBR Green Premix kit (Vazyme) on a
StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA,
USA). The relative expression of each gene was determined and normalized
to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
and calculated by using the 2\textsuperscript{-[?][?]Ct} method.

\textbf{Western blotting}

Mouse splenocytes were lysed and proteins were extracted and blotted
with signal transducer and activator of transcription-3 (STAT3)
(1:1000), pSTAT3 (1:500), and \selectlanguage{greek}β-\selectlanguage{english}actin (1:2000) antibodies (Affinity
Biosciences, USA). Proteins were detected with Super Signal West Pico
chemiluminescence Substrate solution (Thermo Fisher Scientific).
Immuno-reactive bands were visualized by using a gel documentation
system (Syngene, USA). The intensity blots were quantified by
densitometry using Image J software.

\textbf{Cell cultures}

Splenocytes were placed in 48-well tissue culture plates at a density of
5\selectlanguage{ngerman}×10\textsuperscript{5} cells/well with or without SBI (0, 20, 50, 100,
200\selectlanguage{greek}μ\selectlanguage{english}M) (dissolved in DMSO), and the control was a DMSO solution without
SBI. After incubation for 24h at 37 in 5\% CO\textsubscript{2}, the
supernatant and the cells were collected for immediate assay or stored
at -80. To verify the effect of MSCs on mice splenocytes, they were
placed in 24-well tissue culture plates at a density of
6\selectlanguage{ngerman}×10\textsuperscript{4} cells/well with or without SBI (100\selectlanguage{greek}μ\selectlanguage{english}M) and
incubated at 37 \selectlanguage{ngerman}°C with 5\% CO\textsubscript{2} for 4 hours, and then
5×10\textsuperscript{5} splenocytes were added. 24 hours later, the
supernatant and cells were collected for immediate assay or stored at
-80\selectlanguage{english}. To confirm the impact of interleukin (IL) -6, STAT3 and mammalian
target of rapamycin (mTOR) pathways on SBI efficacy, STAT3 agonist
colivelin (MCE, USA) (50nM) and inhibitor Stattic (MCE, USA) (20\selectlanguage{greek}μ\selectlanguage{english}M),
mTOR agonist MHY1485 (MCE, USA) (100nM) and inhibitor Rapamycin (MCE,
USA) (100nM) as well as IL-6 (Genscript BioTECH, USA) (20 ng/ml) were
added into mouse splenocyte cultures respectively with or without SBI
and incubated for 24 hours. The above stimulant concentrations were
provided as a reference by MCE Biotechnology and were used after
\emph{in vitro} validation.

\textbf{Statistical analysis}

All calculations were performed using GraphPad Prism 9.0 software. Data
were presented as mean \selectlanguage{ngerman}± standard error of the mean (SEM). T-tests were
used to assess differences between the two groups. ANOVA followed by
Bonferroni posthoc test was used for multiple comparisons. P value
\textless{} 0.05 was considered significant.

\textbf{Results}

\textbf{SBI alleviated lupus-like disease in} \textbf{R848-induced
mice.}

At 12 weeks, the survival rates of mice in the R848-induced group and
the acetone- treated group were 66.7\% and 100\% respectively (p =
0.065) (Fig. 1A). In comparison to the control group, repeated treatment
of B6 mice with R848 resulted in the development of lupus-like
manifestations, such as splenomegaly, elevated levels of total IgG, ANA,
anti-dsDNA, urine protein, urine creatinine, and serum creatinine (Fig.
1B-I), as well as increased infiltration of leukocytes and deposition of
IgG/C3 in kidney sections (Fig. 1J).

In contrast, the survival rate of all mice induced with R848 and treated
with SBI remained at 100\% after 12 weeks (Fig. 1A). The administration
of SBI exhibited a significant inhibitory effect on spleen enlargement
and resulted in reduced levels of serum IgG, anti-dsDNA, urine protein,
and urine creatinine in R848-induced mice (Fig. 1B-I). Additionally, SBI
treatment demonstrated an improvement in renal pathology, a reduction in
kidney inflammation, and a decrease in the sedimentation of IgG and C3
in the kidney (Fig. 1J), providing evidence that SBI effectively
ameliorates lupus-like symptoms in mouse models.

\textbf{SBI enhanced MSC efficacy in treating R848-induced mice.}

Our data showed that the efficacy of SBI was comparable to that of MSCs,
as evidenced by the inhibition of spleen enlargement and the reduction
of serum IgG, anti-dsDNA, urine protein levels, and kidney IgG and C3
deposition (Fig. 1). There was one mouse in the SBI plus MSCs treatment
group died of cellular embolism during tail vein infusion of MSCs.
Furthermore, the addition of SBI to MSCs treatment resulted in an
enhanced the ability of MSCs to inhibit splenomegaly and serum ANA
levels (Fig. 1B,D). Additionally, SBI increased the extent to which MSCs
improved urine creatinine and serum urea nitrogen levels (Fig. 1G,H),
suggesting a synergistic effect of SBI with MSCs in the treatment of
R848-induced mice.

\textbf{SBI synergized with MSCs to restore immune balance.}

In order to examine the impact of SBI and MSCs on lymphocytes, the
proportions of major T and B cell subsets was analyzed by flow cytometry
(Fig. 2A-C). In comparison to mice treated with acetone, the percentages
of T helper-1 (Th1) cells
(CD4\textsuperscript{+}INF-\selectlanguage{greek}γ\selectlanguage{english}\textsuperscript{+}), T helper-2 (Th2) cells
(CD4\textsuperscript{+}IL-4\textsuperscript{+}), T helper-17 (Th17)
cells (CD4\textsuperscript{+}IL-17\textsuperscript{+}), Tfh cells
(CD4\textsuperscript{+}CXCR5\textsuperscript{+}PD-1\textsuperscript{+}),
germinal center (GC) B cells
(B220\textsuperscript{+}CD95\textsuperscript{+}GL7\textsuperscript{+})
and plasmablast (B220\textsuperscript{+}CD138\textsuperscript{-}) in the
splenocytes of R848-induced mice exhibited an increase, while the
proportions of Treg cells
(CD4\textsuperscript{+}CD25\textsuperscript{+}Foxp3\textsuperscript{+})
and memory B cells
(B220\textsuperscript{+}IgD\textsuperscript{+}CD38\textsuperscript{+})
demonstrated a decrease. The administration of SBI resulted in a
significant reduction in the proportion of Tfh cells and an increase in
the proportion of Treg cells. Consequently, the percentage of
plasmablast cells in splenocytes of mice induced with R848 was also
decreased following SBI treatment (Fig. 2D). Compared to MSCs, combined
treatment with SBI lowed the percentages of Th1 and Th2 cells, decreased
the percentage of Tfh cells (Fig. 2), and further improved the imbalance
of Tfh/Treg cells (Fig. 2). Meanwhile, the absolute number of Tfh cells
was significantly decreased after SBI plus MSCs treatment (Fig. S1),
supporting that SBI could act synergistically with MSCs to rebalance the
immune system.

\textbf{SBI regulated Tfh cell proportion in a dose dependent manner.}

In order to validate the \emph{in vivo} findings of SBI on lymphocytes,
we measured the changes of T and B cell subsets within R848-induced mice
splenocytes cultures subsequent to SBI treatment. Likewise, SBI
treatment successfully rectified the anomalous proportions of Treg
cells, Tfh cells and plasmablast of R848-induced mice \emph{in vitro} ,
yet had no effect on the percentages of Th1 and Th2 cells (Fig. 3A).
These results indicate that SBI primarily participates in the regulation
of Tfh/Treg cell equilibrium. Moreover, our data showed that SBI
inhibited the frequency of Tfh cells in a dose dependent manner. With
the increase of SBI concentration (0- 400\selectlanguage{greek}μ\selectlanguage{english}M), the inhibitory ability of
SBI on Tfh cells gradually increased (Fig. 3B).

\textbf{SBI coordinated with MSCs to modulate IL-6/pSTAT3 signaling.}

In the current and previous studies\textsuperscript{{[}6{]}}, we
revealed that Tfh cells were also an important cell type under MSC
regulation. To explore the mechanism of SBI in collaboration with MSCs
to inhibit Tfh cells, we examined the proliferation and apoptotic status
of Tfh cells in splenocytes of R848-induced mice after different
treatments \emph{in vitro} , and found that both treatments had no
effect on the Ki67\textsuperscript{+} Tfh cell proportion, while the
effect on the Annexin V\textsuperscript{+}Tfh cell proportion was quite
opposite (Fig. 4A).

Next the expression levels of genes closely related to Tfh cell
differentiation were examined by qRT-PCR. Compared with acetone-treated
mice, mRNA expression of inducible T cell costimulator~(\emph{Icos} ),
\emph{Stat3} and \emph{IL-6} was significantly increased in the
splenocytes of R848-induced mice, while SBI treatment reduced mRNA
expression of \emph{Stat3} and \emph{IL-6}(Fig. 4B). SBI enhanced the
ability of MSCs to suppress mRNA expression of \emph{Icos} ,
\emph{Stat3} , and \emph{IL-6} in the splenocytes of R848-induced mice.
(Fig. 4C)\emph{.} Consistently, SBI inhibited the phosphorylation of
STAT3 protein in the splenocytes of R848-induced lupus mice (Fig. 4D),
implying that SBI may coordinate with MSCs to modulate IL-6/pSTAT3
signaling, thereby inhibiting Tfh cell differentiation.

\textbf{IL-6 and its downstream pathways counteracted the effect of SBI
on Tfh cell production.}

To confirm that SBI regulated Tfh cell production through the inhibition
of IL-6, we added IL-6 to R848-induced mice splenocyte cultures with the
presence of SBI. As expected, IL-6 blocked the effect of SBI on Tfh cell
expansion (Fig. 5A). Since IL-6 may initiate downstream signaling
through the mTOR and STAT3 pathways, we subsequently measured the mean
fluorescence intensity of the mTOR downstream targets phosphorylated
serine/threonine kinase Akt (pAKT) and phosphorylated ribosomal protein
S6 (pS6), as well as pSTAT3, by flow cytometry and found that their
expression in Tfh cells was all inhibited by SBI and rebounded upon
addition of IL-6 (Fig. 5B-C). Correspondingly, both the mTOR inhibitor
rapamycin and the STAT3 inhibitor Stattic partially dampened
IL-6-mediated Tfh cell expansion (Fig. 5D-E), whereas both the STAT3
agonist colivelin and the mTOR agonist MHY1485 partially attenuated the
inhibitory effect of SBI on Tfh cells, with colivelin showing a more
pronounced effect compared to MHY1485 (Fig. 5F). These findings suggest
that that SBI inhibits Tfh cell production by regulating IL-6 and its
downstream pathways.

\textbf{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\textsuperscript{{[}}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\textsuperscript{{]}}. 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)\textsuperscript{{[}}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\textsuperscript{{]}}.
Our\emph{in vivo} and \emph{in vitro}study 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\textsuperscript{{[}}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\textsuperscript{{]}}.
Consistent with literature reports\textsuperscript{{[}}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\textsuperscript{{]}}, 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)\textsuperscript{{[}}55{[}{]} Heinrich PC, Behrmann I, Haan S,
Hermanns HM, M\selectlanguage{ngerman}üller-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\textsuperscript{{]}}. 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\textsuperscript{{[}}66{[}{]} Wang, Chaohui et al. ``Protection
by silibinin against experimental ischemic stroke: up-regulated pAkt,
pmTOR, HIF-1\selectlanguage{greek}α \selectlanguage{english}and Bcl-2, down-regulated Bax, NF-\selectlanguage{greek}κ\selectlanguage{english}B
expression.''~Neuroscience letters~vol. 529,1 (2012): 45-50.
doi:10.1016/j.neulet.2012.08.078\textsuperscript{{]}}, and another
research on LPS stimulated porcine mammary epithelial cells showed SBI
increased the expression of mTOR and S6\textsuperscript{{[}}77{[}{]} Xu,
Shengyu et al. ``Silibinin Alleviates Lipopolysaccharide Induced
Inflammation in Porcine Mammary Epithelial Cells via mTOR/NF-\selectlanguage{greek}κ\selectlanguage{english}B
Signaling Pathway.''~Molecular nutrition \& food research~vol. 67,14
(2023): e2200715. doi:10.1002/mnfr.202200715\textsuperscript{{]}}. 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\textsuperscript{{[}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\textsuperscript{{]}}, 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\textsuperscript{{[}}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\textsuperscript{{]}}. 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.

\textbf{Ethics statement}

This study was carried out by the recommendations of the Ethical
Research Committee for Laboratory Animal Welfare of Drum Tower Hospital
of Nanjing University Medical School. The protocol was approved by the
ethical review of Drum Tower Hospital, Nanjing University Medical
School.

\textbf{Author contributions}

NZ, MA contributed to the majority of experiments and data analysis. NZ
drafted the manuscript. YZ, WL, and MH were involved in the analysis of
cellular experiments. TA and YC were involved in the feeding of
experimental animals. XT was involved in experimental teaching and
manuscript editing. YX contributed to the conceptualization of this
study. XF revised the manuscript and was responsible for conceptualizing
and finalizing this study. All authors read and approved the final
manuscript.

\textbf{Conflict of Interest}

The authors declare no conflict of interest.

\textbf{Acknowledgements}

This work is supported by National Key Research and Development Program
of China (2019YFE0111700) and National Natural Science Foundation of
China (81971517). XY was funded by the Science and Technology
Development Fund, Macau SAR (File no. 0075/2019/AMJ).

\textbf{References}

\textbf{Figure legends}

\textbf{Fig. 1} Silybin ameliorated lupus-like features and improved the
efficacy of MSCs in R848-induced mice. (\textbf{A} ) The survival rate
for 12 weeks old acetone-induced B6 mice, R848-induced B6 mice, and
R848-induced mice treated with silybin, MSCs, and silybin plus MSCs (n
=6 in B6 group, n =9 in the rest four groups). (\textbf{B} ) Spleen size
of mice among five groups. (\textbf{C-E} ) Serum IgG, antinuclear
antibody and anti-ds-DNA antibody levels among five groups.
(\textbf{F-I} ) Urine protein, urine creatinine, serum nitrogen and
serum creatinine levels among five groups. (\textbf{J} ) Representative
renal histology and immunofluorescence among five groups (Magnification:
\selectlanguage{ngerman}×400). *P\textless{}0.05, **P\textless{}0.01, ***P\textless{}0.001,
****P\textless{}0.00001, ns: not significant.

\textbf{Fig. 2} Changes in T/B lymphocyte subsets \emph{in vivo} after
silybin and MSCs treatment. (\textbf{A} ) Representative flow cytometry
plots of Th1 cell (CD4\textsuperscript{+}INF-\selectlanguage{greek}γ\selectlanguage{english}\textsuperscript{+}), Th2
cell (CD4\textsuperscript{+}IL-4\textsuperscript{+}) and Th17 cell
(CD4\textsuperscript{+}IL-17\textsuperscript{+}) percentages in
splenocytes among five groups. (\textbf{B} ) Representative flow
cytometry plots of Tfh cell
(CD4\textsuperscript{+}CXCR5\textsuperscript{+}PD-1\textsuperscript{+})
and Treg cell
(CD4\textsuperscript{+}CD25\textsuperscript{+}Foxp3\textsuperscript{+})
percentages in splenocytes among five groups. (\textbf{C} )
Representative flow cytometry plots of memory B cell
(B220\textsuperscript{+}IgD\textsuperscript{+}CD38\textsuperscript{+}),
GC B cell
(B220\textsuperscript{+}CD95\textsuperscript{+}GL7\textsuperscript{+})
and plasmablast (B220\textsuperscript{+}CD138\textsuperscript{+})
percentages in splenocytes among five groups. (\textbf{D} ) Statistical
results of flow cytometry data for each group. n~=~6 in B6 and R848
groups, n~=~9 in silybin or MSCs treated groups, and n~=~8 in silybin
plus MSCs group after removal of dead mice. *P\textless{}0.05,
**P\textless{}0.01, ***P\textless{}0.001, ****P\textless{}0.00001, ns:
not significant.

\textbf{Fig. 3} Effects of silybin on lymphocyte subsets \emph{in vitro}
. (\textbf{A} ) The cell subset proportions were determined by flow
cytometry using splenocytes from two groups (n = 6 for Th1, Th2, Th17,
Treg and Tfh cells, n= 5 for GC B cells and plasmablast). (\textbf{B} )
The percentages of Tfh cells of R848-induced mice treated with different
concentrations of silybin \emph{in vitro} (n=5). *P\textless{}0.05,
**P\textless{}0.01, ***P\textless{}0.001, ****P\textless{}0.00001, ns:
not significant.

\textbf{Fig. 4} Regulatory effects of silybin and MSCs on Tfh cell
proliferation, apoptosis and differentiation-related genes. (\textbf{A}
) The percentages of apoptosis (n=5) and proliferation (n=6) of Tfh
cells in R848-induced mice treated with silybin and MSCs \emph{in vitro}
. (\textbf{B} ) Expression of Tfh-related genes in acetone-induced B6
mice and R848-induced B6 mice (n=6). (\textbf{C} ) Expression of
Tfh-related genes in R848-induced B6 mice and R848-induced mice treated
with silybin and/or MSCs (n=6). (\textbf{D} ) Western blot analysis of
p-STAT3, STAT3 and \selectlanguage{greek}β-\selectlanguage{english}actin in splenocytes of B6, R848-induced and
silybin treated R848-induced mice. *P\textless{}0.05,
**P\textless{}0.01, ***P\textless{}0.001, ****P\textless{}0.00001, ns:
not significant.

\textbf{Fig. 5} Impact of IL-6 and its downstream pathways on silybin
regulated Tfh cells. (\textbf{A} ) IL-6 counteracted the effect of
silybin on Tfh cell expansion in splenocytes of R848-induced mice
(n=6)\emph{.} (\textbf{B} ) The mean fluorescence intensity of pAKT and
pS6 in Tfh cells of R848-induced mice increased after IL-6 treatment
(n=6). (\textbf{C} ) The mean fluorescence intensity of pSTAT3 in Tfh
cells of R848-induced mice increased after IL-6 treatment (n=6).
(\textbf{D-E} ) Both rapamycin and stattic partially inhibited the
effect of IL-6 on Tfh cell expansion in normal B6 mice splenocytes
(n=6). (\textbf{F} ) Both colivelin and MHY1485 counteracted the effect
of silybin on Tfh cell expansion in splenocytes of R848-induced mice
(n=6). *P\textless{}0.05, **P\textless{}0.01, ***P\textless{}0.001,
****P\textless{}0.00001.

\textbf{Hosted file}

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