Effects of Bu Shen Yi Sui Capsule on Th17/Treg cytokines in C57BL/6 mice with experimental autoimmune encephalomyelitis

Background T helper (Th) 17 and regulatory T (Treg) cells play a critical role in the pathogenesis of multiple sclerosis (MS) disease. Bu Shen Yi Sui Capsule (BSYSC), a traditional Chinese medicine formula, has been used clinically for the treatment of MS patients in China. Methods To evaluate the neuroprotective effects and the underlying mechanisms of BSYSC on MS, experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice was induced with myelin oligodendrocyte glycoprotein (MOG) 35–55. Th17 and Treg cells and the related cytokines were detected by flow cytometry, ELISA, real-time quantitative reverse transcription PCR, western blot and immunohistochemistry. Results We found that BSYSC improved neurological function, reduced inflammatory cell infiltration and damage to the axons and myelin in the brain and spinal cord. BSYSC down-regulated markedly the ratio of CD4 + IL-17+/CD4 + CD25 + FoxP3+ T cells in the spleen, decreased the cytokines of IL-17A, IL-6, IL-23, TGF-beta1 in the brain, and dropped the ratio of IL-17A and FoxP3 mRNA and protein in the brain or spinal cord at different stages. Conclusions The study demonstrated that BSYSC had a strong neuroprotective effect on EAE mice. The protective mechanisms of BSYSC might be associated with mediating the regulation of Th17/Treg cells.


Background
Multiple sclerosis (MS) as an inflammatory demyelination disease in the central nervous system (CNS) is pathologically characterized by multifocal inflammation, demyelination, and neuronal damage [1]. Experimental autoimmune encephalomyelitis (EAE), has similar pathological and clinical manifestations to human MS, is the optimal animal model of this disease [2].
A large number of studies have shown the autoimmune responses of MS or EAE are caused mainly by CD4+ T cells and the generation of a T helper (Th) 1/Th2 imbalance [3]. Recent researches have indicated the Th17/regulatory T (Treg) imbalance is a more critical factor in the mechanism underlying the neurological dysfunction of MS/EAE [4]. Th17 cells are a subpopulation of CD4+ T cells that mediate inflammatory responses, while Treg cells are a type of negative immune regulatory cells that mediate immune tolerance. The interactions between Th17 and Treg cells are regulated by the secretion of numbers of cytokines such as interleukin (IL)-17 and transforming growth factor (TGF)-β1, and specific transcription factors, including retinoic acid-related orphan receptor (ROR) γt and forkhead box P3 (FoxP3) [5][6][7]. The pro-inflammatory cytokines attack the myelin and axon, consequently, demyelination and axonal injury occur and result in the onset of MS/EAE [8].
Current therapeutic medicines for MS include corticosteroids and immunosuppressors, such as interferon, glatiramer acetate and natalizumab, show positive effects in inhibiting inflammation since symptoms onset [9]. Thus, prednisone acetate (PA) was selected as the control drug in the present study. Nevertheless, these therapies have potentially side-effects [1]. Traditional Chinese medicine (TCM) can be used to treat the complex and varied presentations of MS, with few side-effects [10]. Bu Shen Yi Sui Capsule (BSYSC, originally named Erhuang Capsule) is a yin-nourishing, phlegm-resolving and blood-activating formula of TCM. The previous clinical studies showed BSYSC had the ability to markedly reduce and eliminate symptoms such as limbs weakness and paresthesia, reduce the frequency and intensity of relapses, ameliorate side-effects of PA and reduces the dose of medication required, to improve the qualities of life in MS patients [11,12]. And it has been approved by the Beijing Food and Drug Administration as a hospital preparation (No. 10003).
Our previous studies have demonstrated BSYSC can regulate Interferon (IFN) -γ and IL-4 production to balance Th1 and Th2 in the serum, brain and spinal cord of EAE rats [13][14][15]. The aim of this study was to explore the regulatory effects of BSYSC on Th17 and Treg cells. Results showed BSYSC had a strong neuroprotective effect on EAE mice, which might be associated with the regulation of Th17/Treg cells.

Medicines and reagents
PA was purchased from Zhejiang Xianju Pharmaceutical Co., Ltd. (Zhejiang, China). Myelin oligodendrocyte glycoprotein peptide (MOG) 35 Bulbus Fritillariae Thunbergii ground into fine powders. The other nine Chinese herbs were extracted twice with boiling water (2 h per extraction, with a total 18-fold volume of purified water). The solutions were concentrated under reduced pressure at 70°C into powders, which were mixed with powders of Bulbus Fritillariae Thunbergii. The resulting mixture of powders was encapsulated. To ensure the quality and stability of BSYSC, the active ingredients were identified by ultra-performance liquid chromatographyquadrupole-time-of-flight-mass spectrometry (UPLC-QTOF-MS). Full-scan spectra were acquired in the mass range m/z 100-1,500 m/z. The compounds identified were loganic acid, decaffeoyl verbascoside, acteoside, isoacteoside, forsythiaside, kaempferol-3-O-α-L-rhamnoside, isorhamnetin-3-O-rutinoside and quercetin-3,3'-dimethyl ether were shown in Figure 1, Table 1, respectively.

Quantification of Acteoside and Forsythiasidein in BSYSC
The powder was ultrasonicated (300 W, 40 kHz) in water for 30 min followed by C18 column chromatography. The samples were analyzed by UPLC-QTOF-MS. The contents of the chief components in BSYSC, acteoside and forsythiaside (derived from Radix Rehmanniae), were 0.79 mg/g and 0.63 mg/g, respectively.

Model establishment and experimental treatment
The mice were randomly divided into four groups: normal control (NC, n = 25), EAE model (EAE, n = 25), PA-treated (n = 25) and BSYSC-treated (n = 25). EAE mice were injected subcutaneously (s.c.) with 0.2 mL emulsion containing 50 μg MOG  in 100 μL of CFA and 100 μL of normal saline (NS), followed by intraperitoneal (i.p.) injections of 500 ng of PTX on Day 0 and Day 2 post-induction (PI) [16]. The 3.02 g/kg dose of BSYSC was shown to be more effective against EAE in our previous study [17]. In the study, the mice in the BSYSC-treated EAE group were given oral suspensions of 3.02 g/kg BSYSC once a day for 40 days. The PA-treated EAE mice were administered PA at a dose of 6 mg/kg. NC and EAE mice were treated with NS. Neurological function scores were based on the sum of the disease state for the tail and all four limbs as follows: 0, no signs of disease in the tail and limbs; scores for the tail were assigned as 1 for paralyzed tail tipparalysis and 2 for tail flaccidity; scores for the limbs were assigned as 1 for gait disturbance, 2 for moderate paralysis and limb dragging and 3 for total paralysis. Mortality was assigned a score of 15 [18]. The grip test records down time from glass bar, which wrapped round gauze to avoid slip of mice. The grip strength test scores were defined as: 0 for 0-4 s, 1 for 5-9 s, 2 for 10-14 s, 3 for 15-19 s, 4 for 20-24 s, 5 for 25-29 s, and 6 for more than 30 s [19].

Sample collection
The mice were sacrificed on Day 6 (early stage, no neurological signs), Day 18 (acute stage, neurological function scores at a peak) and Day 40 (remission stage, no further increase in the signs of EAE) PI. The spleen of four mice in each group were removed and prepared for flow cytometric (FCM) analysis; the brain and spinal cord were immediately frozen for ELISA, qRT-PCR and WB analyses; and 4% paraformaldehyde was used to fix the brain and spinal cord for hematoxylin-eosin (H&E) staining, transmission electron microscopy (TEM) and immunohistochemical (IHC) analysis.

Histopathology and ultrastructural pathology
The brain and spinal cord were fixed, embedded in paraffin, sectioned (thickness, 3 μm) for H&E staining. The sections were observed with light microscopy (Nikon Eclipse 80i, Tokyo, Japan). The inflammatory cell infiltration was scored as follows: 0, no infiltrate; 1, scattered inflammatory cells; 2, single inflammatory cells around blood vessels; 3, inflammatory cell infiltration surrounding blood vessels; 4, inflammatory cell infiltration and perivascular cuff formation, or parenchymal necrosis [20,21]. Cross-sections (1 mm) of the intumescentia lumbalis of the brain and spinal cord were embedded in Epon and sectioned on a Leica EM ultramicrotome. Thin sections (thickness, 70 nm) were viewed on a TEM (JEM-1230, Japan) using a digital camera system to obtain micrographs. Ten high-power fields (×10,000) were selected in the brain (white matter) and spinal cords in each group. The images were analyzed by Image-Pro Plus 6.0 (Media Cybernetics, Inc., Washington, USA) and the results were expressed as area/diameter (mean) of myelin [22][23][24].

Intracellular cytokine analysis by FCM
A single cell suspension of spleen mononuclear cells was first permeabilized with fixation/permeabilzation solution for 30 min at 4°C, washed in perm/wash buffer, and then incubated with mouse anti-CD4 APC, mouse anti-CD25 FITC and mouse anti-FoxP3 PE for 60 min at 4°C. Alternatively, spleen mononuclear cells were incubated with mouse anti-CD4 APC, mouse anti-CD25 FITC and mouse anti-IL-17A PERCP-CY5.5 for 5 hours at 37°C. A control group treated with isotype control antibody was prepared. Cells were fixed in formaldehyde and then analyzed on a BD-FACS Calibur (USA). Data were analyzed based on the percentages of Th17 and Treg cells.

Cytokine measurements by ELISA
The brain of each mouse was weighed and homogenized with cold NS and centrifuged at 3,000 rpm for 20 min to obtain supernatants for cytokine ELISA conducted in strict accordance with instructions provided by the manufacturers. Cytokine (IL-17A, IL-6, IL-23 and TGF-β1) concentrations were determined using the relevant standard curves.
mRNA Analysis by qRT-PCR Total RNA was isolated from approximately 30 mg brain or spinal cord of mice according to the manufacturer's instructions. RNA samples with an OD 260 /OD 280 ratio of 1.9-2.1 and an OD 260 /OD 230 ratio greater than 2.0 were used for the analysis. The cDNA was synthesized from total RNA by the reverse transcription of 1 μg of total RNA using the reverse transcription kit. The primer sequences for PCR designed by the primer premier 5.0 software, based on the GenBank sequences were as follows: FoxP3 (NM_001199347.1) Forward (F), 5'-CTGCCTTGGTACATTCGTGAAC-3', FoxP3 Reverse

Statistical analysis
The data were expressed as mean ± standard error (SE) and analyzed with SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). All the data were firstly subjected to descriptive statistics for normality. The data with normally distributed and equal variances were examined using one-way ANOVA with a post-hoc LSD test, otherwise, the data were performed with a rank-sum test. The family-wise error rate was controlled by the statistical method of Bonferroni. A value of p < 0.05 was considered to indicate statistical significance.

Neurological function and grip test scores
Symptoms of EAE including flaccid tail, staggering gait, hind-limb paralysis, four-limb paralysis and even death appeared sequentially in experimental mice from Day 8 PI. The neurological function scores of EAE mice reached a peak on Day 16. These scores were decreased significantly on Day 17 PI, and from Day 20 to Day 40 PI in BSYSC-treated mice, compared to the scores in EAE mice (p < 0.05, p < 0.01, p < 0.001, respectively; Figure 2). The grip strength test scores of EAE mice appeared primarily from Day 9 PI, with the falling time for EAE mice reduced to less than 30 seconds and the scores reaching a nadir on Day 14 PI. Compared to EAE mice, the scores were significantly higher in BSYSC-treated EAE mice from Day 11 to Day 30 PI (p < 0.01 or p < 0.001, respectively; Figure 2).

Pathological changes on brain and spinal cord tissues
The pathological changes in the brain and spinal cord of mice were observed by light microscopic evaluation of H&E stained tissues on Day 18 and Day 40 PI. The neuronal structure was normal in the NC group. A large number of inflammatory cells were aggregated around small blood vessels to form "sleeve-like" structures in the EAE model group. Conversely, treatment with PA or BSYSC reduced inflammatory cell infiltration. The type of inflammatory cells was classified by lymphocytes, monocytes, neutrophils and eosinophils. The histopathology scores of EAE mice were dramatically increased compared to those of NC mice on Day 18 and Day 40 PI, while treatment with PA or BSYSC significantly reduced the scores (p < 0.001; Figure 3). In further studies, the ultrastructure of the myelin sheath and axons in the brain and spinal cord was observed by TEM. While these were found to be normal in NC mice, a fluffy layer, axonal edema and disintegration were observed in the EAE mice. Demyelination and axonal damage was ameliorated in the PA-and BSYSC-treated EAE mice. The damage was quantified with the ratio of area/diameter (mean) of myelin. The results showed that the ratios of in the brain and spinal cord of EAE mice were markedly increased compared to those of NC mice on Day 18 and 40 PI (p < 0.05 or p < 0.001, respectively; Figure 4). The treatment with PA or BSYSC significantly reduced the ratios (p < 0.05, p < 0.01, p < 0.001, respectively; Figure 4).  Figure 5).

ELISA analysis of inflammation-related cytokines in brain tissue
Compared to NC mice, the levels of IL-17A, IL-6, IL-23 and TGF-β1 in the brain of EAE mice were significantly increased at various times PI:  Figure 6).
qRT-PCR analysis of IL-17A and FoxP3 mRNA expression in brain and spinal cord tissue Compared to NC mice, IL-17A mRNA levels were significantly increased in the brain on Day 18 PI (but not at Day 40) and in the spinal cord on Days 18 and 40 PI in EAE mice compared to NC mice (p < 0.05, p < 0.01).
In contrast, the levels were significantly decreased in PA-and BSYSC-treated EAE mice compared to EAE mice in the spinal cord (p < 0.01). FoxP3 mRNA in the spinal cord was significantly decreased in EAE mice compared to NC mice (p < 0.001), however, there were no differences in the levels detected in the brain of the two groups. BSYSC increased FoxP3 mRNA levels in the spinal cord of EAE mice on Days 18 and 40 PI (p < 0.05 and p < 0.001, respectively). Analysis of the ratio of IL-17A/FoxP3 mRNA levels revealed a significant increase in both the brain and spinal cord on Days 18 and 40 PI in EAE mice compared to NC mice (p < 0.05 and p < 0.001, respectively). In contrast, the ratio was significantly decreased in spinal cord on Days 18 and 40 PI and in the brain on Day 40 PI in PA-and BSYSC-treated EAE mice compared to EAE mice (Figure 7).
WB and IHC analysis of IL-17A and FoxP3 proteins in brain and spinal cord tissue WB analysis demonstrated that IL-17A protein expression in the brain was significantly increased in EAE mice compared to NC mice on Days 18 and 40 PI (p < 0.001 and p < 0.05, respectively). In contrast, levels were significantly reduced in the brain and spinal cord on Days 18 and 40 PI in BSYSC-treated EAE mice compared to EAE mice (p < 0.001 and p < 0.01, respectively). In addition, compared to NC mice, FoxP3 protein expression was obviously decreased in the brain of EAE mice on Day 40  PI and in the spinal cord on Day 18 PI (p < 0.01 and p < 0.001, respectively). BSYSC significantly increased FoxP3 protein in the brain and spinal cord of EAE mice on Days 18 and 40 PI (p < 0.01 and p < 0.001, respectively). Furthermore, the IL-17A/FoxP3 ratios were significantly increased in the brain and spinal cord of EAE mice on Days 18 and 40 PI compared to NC mice (p < 0.001 and p < 0.01 and p < 0.05, respectively). In contrast, the ratios were significantly reduced in the brain and spinal cord of BSYSC-treated EAE mice, compared to EAE mice on Days 18 and 40 PI (p < 0.001 and p < 0.01, respectively; Figure 8). IL-17A and FoxP3 protein expressions in the brain and spinal cord were also detected morphologically by IHC. The results showed that the change trends of IL-17A and FoxP3 protein by IHC were similar to WB (Figure 9).

Discussion
The results of this study showed the EAE induced by administration of MOG  was pathologically characterized by mononuclear cells infiltration, cells shrinkage and nucleuses lysogenic, with neurological deficits associating with demyelination occurred in the brain and spinal cord, which are consistent with previous reports [25]. Comparing with EAE, the EAE + BSYSC manifested an improved neurological function, ameliorated inflammation and demyelination in the CNS. Combining with our previous studies [26], these observations suggested that BSYSC had the neuroprotective effects in EAE mice.
To analyze the neuroprotective mechanisms of BSYSC, we first investigated the development of Th17 and Treg responses in the EAE spleen. It is worth noting that the ratio of CD4 + IL-17+ and CD4 + CD25 + FoxP3 + T cells was obviously increased on Day 6 PI in the spleen of EAE mice. It has been reported that the number of IL-17A-secreting PLP-specific CD4+ T cells, measured by enzyme-linked immunospot assay (ELISPOT), increased significantly in the spleen of SJL mice on Day 8 and in the CNS on Day 25 following the induction of EAE by PLP 139-151 administration [27]. It was indicated that the inflammation reaction was initiated in the peripheral immune organs in the early stage, producing cytokines that disrupt the blood brain barrier (BBB) and allowing immune cells to enter the CNS. Thus, CNS damage occurs as a consequence of peripheral immune responses [28]. Further studies demonstrated that the early treatment with Fasudil, a selective Rho kinase (ROCK) inhibitor, resulted in down-regulation of CD4 + IL-17+ T cells in the spleen on Day 9 PI [29]. In our study, BSYSC significantly regulated the balance between the Th17 and Treg T cells in the spleen of EAE mice in the early stage, resulting in a reduction in the inflammatory responses in peripheral immune organs.
Moreover, we found the levels of inflammation-related cytokines, such as IL-17A, IL-6 and IL-23 increased during the entire course of the disease in the brain of EAE mice. IL-17A, the predominant functional member of the IL-17 family, is a pro-inflammatory cytokine produced by Th17 cells [30]. IL-6 is strongly induced by cells of the innate immune system [31]. Studies have shown that the expressions of these two cytokines are significantly increased in MS/EAE [32,33]. IL-23 is an important factor in Th17 differentiation and the development of EAE [34] and studies have shown that IL-23 and IL-17 secreting cells are attacked to the CNS, leading to demyelination [35,36]. Both PA and BSYSC down regulated the expressions of IL-17A, IL-6 and IL-23 at different stage of EAE, especially in the acute stage. TGF-β1 has been reported to stimulate naïve CD4+ T cells to differentiate into Treg cells which also produce TGF-β1 [37]. Although TGF-β1 exerts an antiinflammatory activity via the induction of FoxP3 expression, in combining with IL-6 and IL-23, TGF-β1 stimulates the production of Th17 cells in EAE [38,39]. In the present study, the high TGF-β1 level in the brain can be speculated to the induction of Th17 cell differentiation by the combination of IL-6 and IL-23, resulting in the exacerbation of EAE [40]. This is supported by the observation that BSYSC reduced not only the levels of IL-17A, IL-6 and IL-23, but also of TGF-β1.
Finally, we targeted the regulatory role of BSYSC played on the IL-17A/FoxP3 ratio in the brain and spinal cord in EAE mice. IL-17A acts an encephalitogenic part in the EAE model, with accumulation of Th17 cells and elevation of IL-17A levels observed in the brain and spinal cord of EAE mice [41,42]. Furthermore, mice lacking Treg cells but having MBP-specific effector T cells can develop spontaneous EAE [43]. In previous literature, immune dysfunction is related with the upregulation of Th17 cells and down-regulation of the Treg cells [40,44]. Therefore, the regulation of immunity balance between Th17 and Treg cells is implicated as novel drug targets for the treatment of MS. Recently, the neuroprotective effect against EAE with electroacupuncture (EA) was reported, the regulation of Th1/Th2/Th17/ Treg by EA was observed as well [45]. However, few studies have investigated the effects of formulas in different tissues and during different stages of EAE. In this study, we found that BSYSC regulated the balance between IL-17A and FoxP3 mRNA and protein expression in the CNS of EAE mice.

Conclusion
In this study, BSYSC exerts significant neuroprotective effects against EAE in mice. The mechanism is related to the decreased number of lymphocytes in the peripheral inflammatory system, thereby reducing the entry of cytotoxic T cells into CNS, and mediating regulation of Th17 and Treg cells and the related cytokines in the CNS.
This study provides a basis for further research into the efficacy of BSYSC and its active ingredients in the treatment of MS. However, the formula contained within BSYSC is complicated and the components exert multi-target effects, thus, the other mechanisms by which BSYSC influences disease in EAE mice remain poorly understood and require further investigation.