Bufei Qingyu Granules Inhibit the Development of Systemic Sclerosis via Notch-1/Jagged-2 Signaling Pathway

Systemic sclerosis (SSc) is a rare chronic autoimmune disorder, mainly characterized by skin sclerosis. In this study, Bufei Qingyu Granules (BQG), a Chinese herbal formula, was used to treat SSc. To better understand the effects and molecular mechanisms of BQG, we successfully established a Bleomycin- (BLM-) induced SSc mouse model, and the mice were treated by BQG. Meanwhile, transcriptomic and bioinformatics analyses were conducted on those samples. As a result, we visually showed that BQG ameliorated the overall health of mice, including body weight, spleen, and thymus index. Thus, it also significantly alleviated inflammation presented by Chemokine (C-X-C motif) ligand 2 (Cxcl2), vasculopathy characterized by α-smooth muscle actin (α-SMA), and fibrotic changes elaborated by not only pathological images, but also the hydroxyproline (HYP) content. After testing by transcriptomic analysis, Cxcl2, Synaptosomal-associated protein 25 (Snap25), and Eukaryotic translation initiation factor 3, and subunit J2 (Eif3j2) which were differentially expressed genes, were verified, so that the data were credible. We further found that BQG could regulate Notch signaling pathway by significantly decreasing both mRNA and protein expression levels of Notch-1 and Jagged-2. Hence, this study demonstrated that BQG could ameliorate the sclerotic skin in mice model involved in inflammation, vascular changes, and fibrosis effects, which was partly mediated by Notch signaling pathway.


Introduction
Systemic sclerosis (SSc) is a rare chronic autoimmune rheumatic disease characterized by persistent extensive fibroproliferation of skin and multiple visceral organs [1]. According to the severity and the involved area, the localized scleroderma should be differentiated from the limited cutaneous scleroderma [2]. Although pathogenesis and the underlying molecular mechanism should be fully elucidated, there is no doubt that this can be attributed to three backbones as follows: innate immune, vasculopathy, and fibrosis [3][4][5].
Although treatment of SSc has greatly attracted the scholar' attention, there is currently no curative therapy resistant to the mentioned disorder [6]. In Chinese medicine, as supplement of modern medicine, there is a treasure, which need to be exploited, especially for this complex disease [7][8][9]. Bufei Qingyu Granules (BQG), that is a traditional Chinese medicine (TCM), is composed of Astragalus mongholicus (Huangqi), Salvia miltiorrhiza (Danshen), Angelica sinensis (Danggui), and other seven Chinese herbs on the basis of a certain proportion of composition. Astragalus mongholicus and Angelica sinensis mixture may have antifibrotic effects 2 Evidence-Based Complementary and Alternative Medicine on renal tubulointerstitial fibrosis and nephrotic syndrome [10,11]. Compound Astragalus and Salvia miltiorrhiza extractions would be also beneficial for fibrotic diseases, such as liver fibrosis and hypertrophic scar [12][13][14]. BQG has been developed and extensively used by Jiangsu Province Hospital of TCM (Nanjing, China) for the treatment of SSc and a portion of pulmonary fibrosis, demonstrating to be highly beneficial in clinical practice. In a preliminary evidence, we previously found the utility of alleviating sclerotic skin with appropriate dosage of BQG in Bleomycin-(BLM-) induced mice model [15]. In recent years, transcriptomics and bioinformatics analyses, providing important lines for understanding of genes' regulations and the mechanisms behind them, were extensively applied in TCM-based researches [16,17]. Here, in order to deeply explore the treatment efficacy of BQG, we obtained a relatively reliable material and sample basis, and the high-throughput sequencing and bioinformatics approaches were subsequently carried out to find out differentially expressed genes (DEGs) and analyze the effects of BQG at both gene and protein expression levels. In addition, we aimed to reveal the potential targets and signaling pathways associated with the treatment efficacy of BQG for SSc.

Materials and Methods
. . Ethics Statement. All animal experiments were strictly performed in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH; Bethesda, MD, USA). This study was reviewed and approved by the Ethics Committee of Nanjing Medical University (Nanjing, China).
. . Preparation of BQG Samples. A total of 10 individual BQG components were commercially available in form of solid granules provided by JiangYin TianJiang Pharmaceuticals Co. Ltd. (Jiangsu, China), which were packaged in 0.5-3 g per bag for each herb. The detailed information of each herb granule was presented in Table 1. Based on our preexperiments, all the herb granules were dissolved in distilled water as solvent to make 1.736 g/mL BQG by an ultrasonic bath for 1 h and then stored at 4 ∘ C. In addition, BQG was fully oscillated and mixed before treating mice.
BQG was quantitated and mixed with double volume of pure methanol, and then the solution was under vortex for 1 min and centrifuged at 12,000 rpm for 10 min at 4 ∘ C. The supernatant was filtered through a membrane filter (0.45 m). Gradient dilution of the filtrate was conducted with 50% methanol before ultra-high-performance liquid chromatography combined with quadrupole-timeof-flight mass spectrometry (UHPLC-Q-TOF/MS) analysis.
. . UHPLC-Q-TOF/MS Analysis. This analysis was carried out by using an Agilent 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA) in combination with a Triple TOF 5600 system equipped with an electrospray ionization (ESI) source (AB SCIEX, Framingham, MA, USA). An XTerra5 MS C18 column (2.1 mm × 100 mm, 3.5 m) was used for the purpose of chromatographic separation and its temperature was maintained at 40 ∘ C during the analysis. The mobile phase was composed of 0.1% aqueous formic acid (A) and acetonitrile (B). The gradient elution program could be used for treatment with 5% B for 0-2 min, with 5%-90% B for 2-9 min, and with 90% B for 9-11 min, and posttreatment for 12-14 min. The flow rate was 200 L/min and the injection volume was 10 L. Positive and negative ion modes were operated, respectively, for the analysis. An ESI source was applied with parameters as follows: Ion Spray voltage, 4500 V; ion source gas 1 (N2), 50 Arb; ion source gas 2 (N2), 50 Arb; gas temperature, 550 ∘ C; curtain gas, 35 Arb; declustering potential, 80 V. The mass spectrum was acquired from 60 to 1500 m/z, and the collision energy was 10 eV.
. . Mice. Female BALB/c mice with the age of 6-week-old were purchased from the Experimental Animal Center of Nantong University (Nantong, China) and were maintained in specific pathogen-free mouse colonies with a 12 h light cycle and temperature varying between 25 and 28 ∘ C. Relative humidity was maintained between 50% and 60%. Before the start of study, all the mice were acclimated to laboratory conditions for within one week. Then, all the mice were randomly divided into three groups as follows: control group Evidence-Based Complementary and Alternative Medicine 3 (n=6); BLM group (n=6); BLM + 34.72 g/kg BQG treatment group (n=6, dosage was selected according to a previous study [15]).

. . Mouse Model of SSc and BQG Treatment.
Here, BLM (Hisun-Pfizer Pharmaceuticals Co. Ltd., China) was dissolved in phosphate-buffered saline (PBS) at the concentration of 400 g/mL and sterilized by filtration as well. Mice underwent a subcutaneous and daily injection of 100 L BLM or PBS solution into a single location on the shaved backs of mice with a 0.45 mm needle. The injection was carried out successively for 4 weeks as previously described [18]. Drug intervention was started during BLM or PBS subcutaneous injection. In the case of BLM exposure model, mice were treated with 0.2 mL/10 g BQG via oral gavage. In addition to BLM group and control group, normal vehicle (PBS) was administered with an equal volume in the same manner.
Mice were weighted and sacrificed 24 h after the last dosage. Some organs and the shaved back of skins were resected for further studies after quick freezing in liquid nitrogen, which were then preserved at -80 ∘ C.
. . Overall Health Assessment. Before mice to be sacrificed, the condition of mice and their skin were daily observed. Body weight (g) of mice was measured and noted weekly. After mice were sacrificed, the lung, spleen, and thymus were taken out and index of organs was calculated in each group of mice according to the following formula: index of organ = weight of organ (mg) / body weight (g) * 100%.
. . Histopathological Examination. All skin sections were cut from the paramidline, lower back shaved region. The skin pieces which were fixed in 4% paraformaldehyde for 24 h were embedded in paraffin routinely. A 5 m-thick tissue section was stained with hematoxylin and eosin (H&E) and Masson's trichrome stain. We evaluated dermal thickness, which was defined as the thickness of skin from the dermalepidermal junction to the junction between the dermis and subcutaneous fat [19,20]. The thickness of dermis was calculated from six different randomly selected fields per specimen by using Image J software (NIH, Bethesda, MD, USA). Slides were examined by standard bright-field microscopy (Nikon Ni-U, China) by two pathologists who were single blinded to the experimental group assignment.
. . Determination of Hydroxyproline Content in Skin Tissue. Hydroxyproline (HYP) assay was used to measure collagen contents. Following manufacturer's instructions of HYPdetection kits (Nanjing Jiancheng Biological Engineering Research Institute, Nanjing, China), 6 mm punch biopsy specimens of shaved back skin tissues were hydrolyzed. The supernatants were collected after chain reaction, and the HYP content was quantified by colorimetric analysis at 550 nm (Synergy HT Microplate Reader; BioTek Instruments, Inc., Winooski, VT, USA) in mice of each group.
. . Enzyme-Linked Immunosorbent Assay (ELISA). Chemokine (C-X-C motif) ligand 2 (Cxcl2) levels in mice serum were collected by cardiac puncture before to be sacrificed and were assessed using ELISA kits (Nanjing Jin Yibai Biological Technology, Nanjing, China) in accordance with the manufacturers' instructions. The final results were presented in histogram.
. . Immunostaining. Immunohistochemistry was carried out using antibodies directed against -smooth muscle actin (Proteintech, Rosemont, IL, USA) abiding by the routine protocols as previously described [21]. Then, all slices were examined independently by two investigators in a blinded manner.
. . Total RNA Extraction. Total RNA isolation from skin tissues was executed respectively by using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in each group according to the manufacturer's introductions. The quantity and purity of RNA were measured by using Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA); thus, RNA integrity was verified by agarose gel.

. . Transcriptomic Assay and Bioinformatics Analysis.
High-quality RNA was used for library construction and high-throughput sequencing. RNA sequencing library was carried out using the VAHTSTM mRNA-seq V2 Library prep Kit (Illumina, Chicago, IL, USA) according to the manufacturer's protocols. The library was then sequenced on a Hiseq platform (Illumina, Chicago, IL, USA) by Sangon Biotech Co., Ltd. (Shanghai, China).
Transcriptome analysis was undertaken using mice's reference genome-based reads mapping. Gene expression levels were estimated using Transcripts Per Million (TPM) values. High-throughput sequencing was performed through applying the criteria of |log 2 fold change|>1, P-values<0.05, and at least one group's mean TPM≥5 as DEGs, which was employed for subsequent analysis.

. . Quantitative Reverse Transcription Polymerase Chain
Reaction (RT-qPCR). The process of RNA extraction was carried out as mentioned previously. Here, 1 g of total mRNA was reverse-transcribed into cDNA synthesized with HiS-cript5 II One Step RT-qPCR Probe Kit (Illumina, Chicago, IL, USA). Target genes were analyzed by RT-qPCR according to the manufacturer's instructions (Applied Biosystems Inc., Foster City, CA, USA). The associated primer sequences are listed in Table 2. The relative mRNA expression levels were quantified with the 2 −ΔΔCt method and the amplified transcript level of each specific gene was normalized to the expression of the endogenous control GAPDH.
. . Antibodies and Western Blot Analysis. The radio immunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) was added into skin tissues, while the tube was placed on ice for 40 min. The tissue was then centrifuged at 12000 rpm for 5 min at 4 ∘ C to remove pellet. The lysate, which was transferred to a fresh tube, was measured by using  . . Statistical Analysis. The data were presented as mean ± standard deviation (SD). Data analysis was performed by using GraphPad Prism 6 software, version 6.0a (GraphPad Software, USA). Averaged data of two groups were compared using unpaired two-sample t-test. One-way analysis of variance (ANOVA) with Bonferroni adjustment were used to perform multiple group comparisons. A P-value<0.05 was considered statistically significant.

. . Typical Chemical Components and eir Contents in BQG
Are Identified by UHPLC-Q-TOF/MS. To clarify the material basis of BQG for the treatment efficacy, the representative chemical components in BQG confirmed by UHPLC-Q-TOF/MS were detected under the optimized conditions. As data are shown in Figures 1(a) and 1(b) and Table 3, a total of 14 peak signals and the content of each constituent were identified and calculated, respectively.
. . Effects of BQG Are Showed in Overall Health of Mice. In this experiment, the overall health of mice was observed as a routine. The conditions of mice in the control group treated with PBS, which included the spirit, food intake, activity, and body weight, were much better than the BLM group at the end of the point, and also the hair in shaved area was almost recovered. Compared with the model group, the status of BQG-treated mice exceeded; thus, the degree of dermal sclerosis was lighter, not as the hair around the injection site stopping growing. There was no significant difference in body weight of mice between each group before the start of the experiment (P=0.056). The mice in model group had the lowest body weight among the three groups. BQG-treated group had the higher body weight compared with the model group (19.4983 ± 0.83968 g vs 17.7833 ± 0.94534 g, P=0.004; Figure 2(a)). The organ index, including lung, spleen, and thymus, was used to reflect the status of the animal's function Evidence-Based Complementary and Alternative Medicine  in a chronic drug experiment, especially for the thymus and spleen as important indicator reflecting the immune function and splenomegaly occurrence of the body to some extent. As illustrated in Figure 2(b), there was no significant difference in lung index between the groups, P>0.05. There was apparently lower level of spleen index in BQG-treated group compared with the model group (P<0.001). However, the level of thymus index obviously increased compared with the model group (P<0.001).
. . BQG Attenuates Skin Sclerosis in BLM-Induced Animal Model. In this section, our eyes were fixed to indicate whether BQG has the valid effects on BLM-induced mice model. To the end, lesional skin sites were thicker in BLMinduced mice than in PBS-treated mice on 28th day, and no effects were observed on the shaved skin of PBS-treated mice. However, dermal thickness was visualized to be obviously decreased by BQG intervention in both slices stained by H&E and Masson's trichrome (Figures 3(a) and 3(b)). Moreover, histogram was used to quantify dermal thickness and levels of HYP content in each group of mice, which showed that both of two items were significantly reduced in BLM exposure mice treated with BQG compared with those which did not receive BQG (Figures 3(c) and 3(d)).
In addition, -smooth muscle actin ( -SMA) expression was evaluated by immunohistochemistry, which showed that proliferation of positive vascular smooth muscle cells visualized as like vessel wall thickness was decreased in BQG group, compared with the BLM group mice (Figure 4). Besides, inflammation involved in this progress was detected by the production levels of Cxcl2. As shown in Figure 5, BQG group cut the expression by almost half percent compared with BLM model group.
Taken together, these results indicated that BQG had a potential inhibition role in inflammation, vascular changes, and fibrosis in the BLM-induced SSc mouse model.

. . DEGs Are Expressed between BLM Model and BQG-Treated Group.
A comparative analysis between the control, BLM, and BQG-treated groups for transcriptome analysis and gene expression was conducted. In total, the RNA-Seq results included within 51826 genes. As displayed in     compared with the control group, 1502 genes were upregulated in the BLM model group, among which 945 genes were changed in the BQG-treated group. In addition, 618 genes were downregulated in the BLM model group, among which 103 genes were reversed in the BQG-treated group. From Figures 6(b) and 6(c), the condition of DEGs, especially between the BLM model and BQG treatment groups, could be observed. By the way, the pathologic changes of sample 3 were not as severe as that another two. Maybe this is the reason why sample 3 of BLM group is a little bit different from another two in heat map. Relatively small sample size may also be a factor to lead to this phenomenon. Thus, adding sample 3 will not significantly improve the original results. The top 10 up-and downregulated DEGs were ranked after intervention of BQG compared with BLM model group, respectively (see Tables 4 and 5).

. . Notch Signaling Serves as a Candidate Pathway in
Regulating SSc. In order to further discover the potential functional pathways variated by BQG, we carried out Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for DEGs, and the top 10 DEGs are listed in Table 6. Notch signaling pathway ranking 5 was priorly chosen to be 8 Evidence-Based Complementary and Alternative Medicine  verified in the following experiments (Figure 7). To explore the biological function of differentially expressed mRNA, Gene Ontology (GO) pathway annotation was conducted. It was revealed that, under the undistinguished circumstance of up-or downregulated relationship, single-organism cellular process was the most significantly enriched pathway for biological processes, compared with the BLM model group. In addition to the analysis of molecular function enrichment, the DEGs were enriched in protein binding. Moreover, intracellular part was the most obvious pathway Evidence-Based Complementary and Alternative Medicine 9  Table 7.
. . Notch-and Jagged-Play e Important Roles in e Pathological Progress of SSc. Based on the above mentioned results, overlapped DEGs were identified as potential targets and further confirmed by RT-qPCR as well. In the results, the trends of Cxcl2, Synaptosomal-associated protein 25 (Snap25), Eukaryotic translation initiation factor 3, subunit J2 (Eif3j2), Notch-1, and Jagged-2 were illustrated in Figures 8(a)-8(e); thus, there was a statistical significance at the transcript expression level between the groups. In addition, Western blot analysis was carried out using the same remaining samples. It is noteworthy that Notch-1 and Jagged-2 (Figures 9(a)-9(c)) were significantly elevated in the BLM model group compared with the control group at the protein expression level. On the contrary, the decrease of the expression level of Notch-1 and Jagged-2 was detected in BQG-treated group. As a result, Notch signaling pathway was associated with proteins named Notch-1 and Jagged-2, which participated in the construction of SSc model and the treatment process of BQG.

Discussion
The purpose of our present study was to achieve the BQG's molecular evidence against dermal sclerosis out of mess and to provide a new evidence for network pharmacology. We showed the representative constitutes and the contents of BQG, and the BQG's preventive effects on SSc were revealed as well. Furthermore, 5 potential targets were sought out and validated in 1048 genes altered after BQG treatment. Thus, the possible mechanism was verified, which inhibited the Notch signaling pathway. BQG, consisting of ten Chinese herbs, is mainly used for treating the SSc's "Lung deficiency generating phlegm stasis" syndrome. In addition, some evidences about its effects associated with fibrosis, which is the footstone of SSc, could be found in previous studies. Danshensu, one of the major components derived from Salvia miltiorrhiza Bunge, can attenuate cardiac fibrosis and hepatic fibrosis [22,23]. Amygdalin can reduce the BLM-induced increase of proteinic biomarkers in rat serum [24]. It also can attenuate kidney fibroblast activation and rat renal interstitial fibrosis [25]. Paeonol has therapeutic functions on BLM-induced pulmonary fibrosis in mice and, at least in part, could be mediated by the inhibition of the MAPKs/Smad3 signaling pathway [26]. As the main active substance of Astragalus membranaceus Bunge, astragaloside IV also contains the potent protective effect on cardiovascular disease, pulmonary disease, and liver fibrosis [27]. Because BQG is a material basis for the treatment efficacy, the main focus in the next step will be to identify and separate precise active ingredients.
However, the effect of compound drugs cannot be replaced by a single component. To uncover the possible mechanism by a holistic approach, the BLM-induced SSc mouse model was previously tested successfully, which was consistent with our present in vivo study. Over time, BQG ameliorated the overall health of mice, including body weight. Thus, BQG-treated group attenuated the level of spleen index and increased thymus index conversely to a certain extent, indicating that BQG could prevent the occurrence of splenomegaly and play a protective role in immune system. Additionally, BQG administration stopped the tendency of skin sclerosis development reflected by not only pathological images, but also the HYP content. Cxcl2 is a chemokine primarily functions with recruiting neutrophils [28,29]. Besides, we found that its expression level increased in model group, which was in agreement with the findings of a previous research [30]. BQG also significantly reduced Cxcl2 level, indicating that BQG alleviated inflammation partly due to Cxcl2 or the involvement of neutrophils. Vasculopathy characterized by -SMA plays an important role in the pathogenesis of SSc [31,32]. BQG was showed to be advantageous to improve the expression level of -SMA-positive cells in vascular walls, and it was revealed that BQG's effect might be possibly related to vascular changes. Previous evidences supported this consequence as well [21,31]. All these efforts provided a reliable sample basis for subsequent analysis. Additionally, we for the first time illustrated the underlying mechanisms of this old traditional formula using transcriptomics and bioinformatics analyses to examine the possible molecular targets affected by BQG. RNA-sequency expression profiling showed that 945 genes were altered after BQG administration. Three predicted target genes were selected for validation by RT-qPCR, whose fold-change tendencies of Cxcl2, Snap25, and Eif3j2 were consistent with transcriptomic data. These results consolidated that RNA-sequencing data were credible. Then, DEGs were analyzed by protein-protein interaction (PPI) network and Notch-1/Jagged-2 signaling was sort out. Furthermore, we confirmed them in gene level by RT-qPCR, and their associated proteins were validated by Western blotting as well.
Notch signaling pathway ranked 5 in our data, and that is a conserved developmental pathway, participating in regulating all kinds of key cellular processes [33], as well as acting in the SSc pathogenesis. Clara et al. demonstrated that Notch signaling can be highly activated in SSc, and it also can promote collagen release and activation of fibroblast isolated from the skin samples. Additionally, fibrosis could be ameliorated by inhibition of Notch signaling pathway, applying a -secretase inhibitor (DAPT) or overexpression of a Notch-1 antisense construct [34,35], which was in agreement with Kavian et al. 's findings [36]. It was reported that Notch deficiency resulted in a crucial inhibitory effect on the response to BLM-induced dermal fibrosis and lung fibrosis [37]. To our knowledge, core elements of this signaling pathway consists of four Notch transmembrane receptors and five transmembrane ligands in mammals named as follows: Notch 1-4, three Delta-like proteins (DLL1, DLL3, and DLL4), and two Jagged proteins (Jag 1, 2). However, the detailed contribution of the diverse Notch receptors has remained obscure, especially in terms of occurring in a variety of circumstances. Two-step proteolysis of the receptors was caused by binding and interaction between Notch receptors and their ligands. Then, an active form of the Notch intracellular domain (NICD) was released and translocated from cytoplasm to nucleus, where it ultimately interacted with transcriptional repressors, stimulating the expression level of various genes [38][39][40]. Several tissues express not only Jagged-2, but also Notch-1 because Notch-1 is a cognate receptor for Jagged-2 [41]. To date, there is no enough evidence on how Notch signaling pathway stimulates collagen release in fibrotic diseases on a molecular level. Here, as schematic diagram shown in Figure 10, we only provided a novel evidence that Notch-1 and Jagged-2 were also elevated in the BLM-induced SSc mouse model. Moreover, depending on BQG that repressed the expression level of Notch-1 and Jagged-2, it is suggested that the reduction of progress in skin sclerosis may be mediated by inhibition of the activation of Notch signaling pathway. Consequently, it could enhance the current understanding of Notch signal transduction in BLM-induced SSc mouse model, although it was not fully elucidated whether BQG acted directly or indirectly.
Nevertheless, some limitations of our study should be pointed out. Firstly, the presented Chinese medicinal formula is pretty complex, especially in the efficacy and mechanism of chemical components which needs to be further explored with more samples size. Secondly, as different mice models have different clinical features and molecular bases, more various mice models should be used to evaluate the effects of BQG on SSc treatment to further support our findings. Thirdly, a small part of target genes was confirmed in this experiment; the rest latently should be under consideration for the sake of moving as far as possible to "Precision Medicine" in Chinese herbs.

Conclusion
The results showed that the administration of BQG can prevent sclerotic skin induced by BLM in mice model, and this process is partly associated with decrease of inflammation, vascular changes, and fibrosis effects, so as to suppress the production of fibrillar collagens, which are modulated by blocking Notch signaling pathway.