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

Authors: Minhui Su; Fang Tian; Bingchen Ouyang; Xiaoyu Wu; Feng Guo; Hua Chen; Xuejun Zhu; Jianmei Chen; Xian Qian
Journal: Evidence-Based Complementary and Alternative Medicine
 (2019)

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## Abstract

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 byα-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.


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## Body

## 1. 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 [3–5].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 [7–9]. Bufei Qingyu Granules (BQG), that is a traditional Chinese medicine (TCM), is composed ofAstragalus mongholicus (Huangqi),Salvia miltiorrhiza (Danshen),Angelica sinensis (Danggui), and other seven Chinese herbs on the basis of a certain proportion of composition.Astragalus mongholicus andAngelica sinensis mixture may have antifibrotic effects on renal tubulointerstitial fibrosis and nephrotic syndrome [10, 11]. CompoundAstragalus andSalvia miltiorrhiza extractions would be also beneficial for fibrotic diseases, such as liver fibrosis and hypertrophic scar [12–14]. 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.


## 2. Materials and Methods

### 2.1. 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).


### 2.2. 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 Table1. 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.Table 1
The batch number and produced date of each herb.
Chinese name Herb (Local name) Medicinal parts Amount (g) Batch number HangQi Astragalus membranaceus (Fisch.) Bge.var. mongholicus (Bge.) Hsiao Root 30 g 1705165 DangShen Codonopsis pilosula (Franch.) Nannf. Root 20 g 1706201 ShanYao Dioscorea opposita Thunb. rhizome 20 g 1705108 DanPi Paeonia suffruticosaAndr. root bark 12 g 17121101 DanShen Salvia miltiorrhiza Bge. Root 20 g 1706030 DangGui Angelica sinensis (oliv.)Diels. Root 10 g 1707182 TaoRen Prunus persica(L) Batch. Seed 10 g 17121371 WuWeizi Schisandra chinensis(Turcz.) Baill. Fruit 6 g 1706066 LingXiaoHua Campsis grandiflora (Thunb.) K. Schum. Flower 10 g 1609126 JieGeng Platycodon grandiforum(Jacq) A. DC. Root 6 g 18011431BQG 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 (0.45μm). Gradient dilution of the filtrate was conducted with 50% methanol before ultra-high-performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) analysis.


### 2.3. 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 XTerra® MS C18 column (2.1 mm × 100 mm, 3.5μm) 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 μL/min and the injection volume was 10 μL. 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.


### 2.4. 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 (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]).


### 2.5. 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 400μg/mL and sterilized by filtration as well. Mice underwent a subcutaneous and daily injection of 100 μL 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.


### 2.6. 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%.


### 2.7. 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 5μm-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 dermal-epidermal 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.


### 2.8. Determination of Hydroxyproline Content in Skin Tissue

Hydroxyproline (HYP) assay was used to measure collagen contents. Following manufacturer’s instructions of HYP-detection 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.


### 2.9. 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.


### 2.10. Immunostaining

Immunohistochemistry was carried out using antibodies directed againstα-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.


### 2.11. 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.


### 2.12. 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|log2 fold change|>1,P-values<0.05, and at least one group’s mean TPM≥5 as DEGs, which was employed for subsequent analysis.


### 2.13. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

The process of RNA extraction was carried out as mentioned previously. Here, 1μg of total mRNA was reverse-transcribed into cDNA synthesized with HiScript® 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.Table 2
Primer sequences for RT-qPCR.
Targets Forward primer (5’-3’) Reverse primer (3’-5’) Mus-Cxcl2 CCAACCACCAGGCTACAGG GCGTCACACTCAAGCTCTG Mus-Snap25 GCTGGAGGAGATGCAGAGGA TCCAGTTGTTCGCCTTGCTC Mus-Eif3j2 ACCAACTCATTGACTGTGCTCTGC CCACCATAATCTGCCAGGTCGTC Mus-Notch1 ATGCTGCTGTTGTGCTCCTGAAG CGGCAATCGGTCCATGTGATCC Mus-Jagged2 AAGGAGTACCAGGCCAAGGTGAC CCGGCGGCAGGTAGAAGGAG Mus-GAPDH CACCATCTTCCAGGAGCGAG CCTTCTCCATGGTGGTGAAGAC


### 2.14. 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 BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Add 5 × SDS stop buffer to the lysate so as to reach 1 × SDS final. Load denatured mixture of protein and protein markers into the gel and transfer onto Polyvinylidene difluoride (PVDF) membranes. After electrotransferring and blocking with 1 × TBST containing 5% nonfat dry milk, membranes were incubated with primary antibodies overnight at 4°C: mouse monoclonal GAPDH (1:10000; Proteintech, Rosemont, IL, USA), rabbit monoclonal Anti-Jagged2 (1:500; Abcam, Cambridge, UK), and Notch1 XP®rabbit mAb (1:500, Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were incubated with the horseradish peroxidase- (HRP-) conjugated secondary antibody diluted at 1:2000 (Proteintech, Rosemont, IL, USA) at room temperature for 1 h. Last but not least is the exposure under a chemiluminescence imaging system (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH was used as a loading control. All the protein bands were analyzed by using Image J software.


### 2.15. 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. AP-value<0.05 was considered statistically significant.


## 2.1. 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).


## 2.2. 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 Table1. 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.Table 1
The batch number and produced date of each herb.
Chinese name Herb (Local name) Medicinal parts Amount (g) Batch number HangQi Astragalus membranaceus (Fisch.) Bge.var. mongholicus (Bge.) Hsiao Root 30 g 1705165 DangShen Codonopsis pilosula (Franch.) Nannf. Root 20 g 1706201 ShanYao Dioscorea opposita Thunb. rhizome 20 g 1705108 DanPi Paeonia suffruticosaAndr. root bark 12 g 17121101 DanShen Salvia miltiorrhiza Bge. Root 20 g 1706030 DangGui Angelica sinensis (oliv.)Diels. Root 10 g 1707182 TaoRen Prunus persica(L) Batch. Seed 10 g 17121371 WuWeizi Schisandra chinensis(Turcz.) Baill. Fruit 6 g 1706066 LingXiaoHua Campsis grandiflora (Thunb.) K. Schum. Flower 10 g 1609126 JieGeng Platycodon grandiforum(Jacq) A. DC. Root 6 g 18011431BQG 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 (0.45μm). Gradient dilution of the filtrate was conducted with 50% methanol before ultra-high-performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) analysis.


## 2.3. 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 XTerra® MS C18 column (2.1 mm × 100 mm, 3.5μm) 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 μL/min and the injection volume was 10 μL. 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.


## 2.4. 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 (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]).


## 2.5. 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 400μg/mL and sterilized by filtration as well. Mice underwent a subcutaneous and daily injection of 100 μL 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.


## 2.6. 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%.


## 2.7. 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 5μm-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 dermal-epidermal 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.


## 2.8. Determination of Hydroxyproline Content in Skin Tissue

Hydroxyproline (HYP) assay was used to measure collagen contents. Following manufacturer’s instructions of HYP-detection 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.


## 2.9. 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.


## 2.10. Immunostaining

Immunohistochemistry was carried out using antibodies directed againstα-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.


## 2.11. 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.


## 2.12. 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|log2 fold change|>1,P-values<0.05, and at least one group’s mean TPM≥5 as DEGs, which was employed for subsequent analysis.


## 2.13. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

The process of RNA extraction was carried out as mentioned previously. Here, 1μg of total mRNA was reverse-transcribed into cDNA synthesized with HiScript® 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.Table 2
Primer sequences for RT-qPCR.
Targets Forward primer (5’-3’) Reverse primer (3’-5’) Mus-Cxcl2 CCAACCACCAGGCTACAGG GCGTCACACTCAAGCTCTG Mus-Snap25 GCTGGAGGAGATGCAGAGGA TCCAGTTGTTCGCCTTGCTC Mus-Eif3j2 ACCAACTCATTGACTGTGCTCTGC CCACCATAATCTGCCAGGTCGTC Mus-Notch1 ATGCTGCTGTTGTGCTCCTGAAG CGGCAATCGGTCCATGTGATCC Mus-Jagged2 AAGGAGTACCAGGCCAAGGTGAC CCGGCGGCAGGTAGAAGGAG Mus-GAPDH CACCATCTTCCAGGAGCGAG CCTTCTCCATGGTGGTGAAGAC


## 2.14. 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 BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Add 5 × SDS stop buffer to the lysate so as to reach 1 × SDS final. Load denatured mixture of protein and protein markers into the gel and transfer onto Polyvinylidene difluoride (PVDF) membranes. After electrotransferring and blocking with 1 × TBST containing 5% nonfat dry milk, membranes were incubated with primary antibodies overnight at 4°C: mouse monoclonal GAPDH (1:10000; Proteintech, Rosemont, IL, USA), rabbit monoclonal Anti-Jagged2 (1:500; Abcam, Cambridge, UK), and Notch1 XP®rabbit mAb (1:500, Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were incubated with the horseradish peroxidase- (HRP-) conjugated secondary antibody diluted at 1:2000 (Proteintech, Rosemont, IL, USA) at room temperature for 1 h. Last but not least is the exposure under a chemiluminescence imaging system (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH was used as a loading control. All the protein bands were analyzed by using Image J software.


## 2.15. 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. AP-value<0.05 was considered statistically significant.


## 3. Results

### 3.1. Typical Chemical Components and Their 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 Figures1(a) and 1(b) and Table 3, a total of 14 peak signals and the content of each constituent were identified and calculated, respectively.Table 3
Identification of typical chemical components and its contents in BQG.
Peak Identification Content (ug/g) Source 1 Amygdalin 2023.5 TaoRen 2 Paeonol 764.5 DanPi 3 Schisandrol A 38.7 WuWeiZi 4 Dihydrotanshinone I 9.3 DanShen 5 Cryptotanshinone 11.5 DanShen 6 Schizandrin A 0.7 WuWeiZi 7 Tanshinone IIA 9.9 DanShen 8 Schizandrin B 0.7 WuWeiZi 9 Danshensu 2290.2 DanShen 10 Acteoside 549.3 LingXiaoHua 11 Ferulic acid 45.2 DangGui 12 Lobetyolin 31.0 DangShen 13 Platycodin D 76.2 JieGeng 14 Astragaloside IV 196.0 HangQiFigure 1
Fingerprints of BQG samples in the positive (a) and negative (b) ion modes by UHPLC-Q-TOF/MS. Peak 1, Amygdalin; Peak 2, Paeonol; Peak 3, Schizandrol A; Peak 4, Dihydrotanshinone I; Peak 5, Cryptotanshinone; Peak 6, Schizandrin A; Peak 7, Tanshinone IIA; Peak 8, Schizandrin B; Peak 9, Danshensu; Peak 10, Acteoside; Peak 11, Ferulic acid; Peak 12, Lobetyolin; Peak 13, Platycodin D; Peak 14, Astragaloside IV.
(a) (b)


### 3.2. 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 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).Figure 2
Overall health assessment after BQG treatment in SSc mouse model. (a) The curves of body weight of mice in different groups at different time points. (b) The percentage of lung, spleen, and thymus index, n=6 per group;∗P<0.05, ∗∗P<0.01 compared with the model group.
(a) (b)


### 3.3. 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 BLM-induced 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 (Figures3(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)).Figure 3
Effects of BQG in improving BLM-induced skin sclerosis of mouse model. Skin sections derived from BLM or PBS-treated mice in the presence or absence of BQG administration were stained with hematoxylin and eosin (a) and Masson’s trichrome (b). Representative images are shown. Scale bar=100μm. (c) Data of dermal thickness are presented by histogram. (d) The levels of HYP content in each group of mice.In addition,α-smooth muscle actin (α-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.Figure 4
α-SMA expression was evaluated by immunohistochemistry. Black arrows indicated α-SMA-positive cells in vascular walls. Representative images are shown. Original magnifications are 200× (scale bar=100 μm) and 400× (scale bar=50 μm), respectively.Figure 5
ELISA assay showed elevated Cxcl2 in the serum derived from mice in different groups. Each graph illustrates mean ± SD of the indicated parameters (n≥3 per group;∗P<0.05, ∗∗P<0.01 vs 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.


### 3.4. 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 Figure6(a), both up- and downregulated DEGs were overlapped. Of these, transcriptomic analysis revealed that, 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).Table 4
Upregulated DEGs after intervention of BQG.
Gene ID Gene Name log2 fold change P-value ENSMUSG00000097656 Gm26712 7.98659314 1.31E-06 ENSMUSG00000105338 Gm43802 7.80359975 3.28E-08 ENSMUSG00000106587 AC125099.2 7.470051892 3.57E-06 ENSMUSG00000103308 Gm37800 4.023628692 4.11E-05 ENSMUSG00000049421 Zfp260 3.689570886 9.22E-06 ENSMUSG00000003545 Fosb 3.68790389 3.35E-06 ENSMUSG00000057465 Saa2 3.572774437 2.45E-06 ENSMUSG00000079025 Gsdmc 3.355515254 8.02E-06 ENSMUSG00000043424 Eif3j2 3.102051328 2.66E-07 ENSMUSG00000046311 Zfp62 2.135549165 1.47E-06Table 5
Downregulated DEGs after intervention of BQG.
Gene ID Gene Name log2 fold change P-value ENSMUSG00000027273 Snap25 -6.26729 1.36E-05 ENSMUSG00000084257 Gm11597 -6.26702 8.33E-05 ENSMUSG00000083405 Gm15725 -5.90441 6.95E-07 ENSMUSG00000102564 Gm37035 -5.4123 6.22E-08 ENSMUSG00000058427 Cxcl2 -5.30387 4.51E-06 ENSMUSG00000097724 Gm26850 -5.06965 1.66E-05 ENSMUSG00000099068 Gm27861 -4.83004 4.91E-05 ENSMUSG00000029379 Cxcl3 -4.54472 8.15E-05 ENSMUSG00000097078 Gm26566 -4.25859 9.63E-05 ENSMUSG00000103309 BC037039 -4.22422 3.50E-05Figure 6
Transcriptomic assay and bioinformatics analysis. (a) Venn diagram of overlapping genes derived from transcriptome analysis in a pairwise comparison. (b) Compared with BLM group, filtering of significant DEGs was presented by valcano. (c) Heat map of regulated DEGs in skin samples of control, BLM, and BQG-treated groups. Red color represents upregulated genes, while green color shows downregulated genes. The depth of color represents difference on multiplier.
(a) (b) (c)


### 3.5. 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 Table6. Notch signaling pathway ranking 5 was priorly chosen to be 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 of cellular component, which had the enriched DEG called Notch-1. Both up- and downregulated mRNAs significantly enriched GO terms, and the top 20 DEGs are presented in Table 7.Table 6
Signaling pathway prediction of DEGs between BQG and BLM group from KEGG database.
ID Description P-value Significant symbol genes UP-REGULATED  ko03010 Ribosome 0.001525 Rpl30, Rpl26, Rpl35a, Rpl31, Rps27a, Rpl27-ps3 ko04924 Renin secretion 0.002328 Calm4, Gucy1a3, Adcyap1r1, Gucy1b3 ko03030 DNA replication 0.004173 Rnaseh2b, Rpa3, Mcm6 ko04624 Toll and Imd signaling pathway 0.004522 Birc3, Tab2, Faf1 ko04713 Circadian entrainment 0.008117 Calm4, Gucy1a3, Adcyap1r1, Gucy1b3 ko05031 Amphetamine addiction 0.018481 Calm4, Fosb, Ppp1cb ko00230 Purine metabolism 0.018499 Nt5e, Entpd5, Gucy1a3, Gucy1b3, Pde4d ko04970 Salivary secretion 0.020157 Calm4, Gucy1a3, Gucy1b3 ko04611 Platelet activation 0.022505 Gucy1a3, Vamp8, Ppp1cb, Gucy1b3 ko04270 Vascular smooth muscle contraction 0.023148 Calm4, Gucy1a3, Ppp1cb, Gucy1b3  DOWN-REGULATED  ko04550 Signaling pathways regulating pluripotency of stem cells 0.001454 Inhbb, Wnt4, Map2k2, Dvl1, Mapk13, Id1, Fgfr3, Axin2, Tcf3, Myf5, Wnt9a, Fzd8, Fzd2, Dlx5, Wnt3a, Fgfr4 ko04360 Axon guidance 0.002752 Wnt4, Sema3f, Sema4g, Efnb1, Plxna3, Ssh3, Epha2, Pak6, Abl1, Pak4, Ephb3, Plxnb1, Efna2, Fes, Nck2, Ephb6, Sema4f, Sema4b ko04115 p53 signaling pathway 0.002898 Sfn, Gtse1, Gadd45g, Gadd45a, Rprm, Gadd45b, Pidd1, Sesn2, Bbc3, Bax ko04390 Hippo signaling pathway 0.003971 Wnt4, Dvl1, Llgl2, Id1, Axin2, Llgl1, Wnt9a, Fzd8, Fzd2, Wwc1, Scrib, Tead3, Ajuba, Wnt3a, Bbc3, Smad7 ko04330 Notch signaling pathway 0.006239 Dvl1, Hes1, Dtx2, Lfng, Jag2, Notch1, Ncor2 ko05166 HTLV-I infection 0.006668 Wnt4, Dvl1, Cdc20, Ranbp3, Ranbp1, Crtc1, Map3k14, Tcf3, Pold1, H2-Q2, Wnt9a, Msx1, Bcl2l1, Fzd8, Fzd2, Cdkn2b, H2-Q6, Ltbr, Wnt3a, Pdgfa, Slc2a1, Mybl2, Bax, Fosl1 ko04014 Ras signaling pathway 0.010254 Ngfr, Map2k2, Pla2g6, Rgl2, Arf6, Gnb2, Fgfr3, Rin1, Epha2, Pak6, Abl1, Pak4, Rasal3, Pla2g4f, Efna2, Bcl2l1, Zap70, Pdgfa, Fgfr4, Rasal1 ko05322 Systemic lupus erythematosus 0.012114 Hist1h2bl, Hist3h2a, Hist1h2br, Hist1h4n, Hist1h4m, H2afz, Hist1h3d, H2afj, Hist1h4k, Hist2h3c2, Hist1h3i, Hist1h2bg, Hist1h2bk, H2afx ko00680 Methane metabolism 0.012152 Shmt2, Eno1b, Pfkl, Phgdh, Eno1 ko05230 Central carbon metabolism in cancer 0.014355 Slc16a3, Map2k2, Fgfr3, Hk3, Pfkl, Sirt6, Erbb2, Slc2a1Table 7
The significantly enriched GO terms of DEGs ranking top 20.
Ontology ID Term Gene count P-value UP-REGULATED  biological process GO: 0008152 metabolic process 119 4.60E-07 GO: 0044237 cellular metabolic process 107 4.20E-06 GO: 0044238 primary metabolic process 108 6.30E-06 GO: 0071704 organic substance metabolic process 111 6.90E-06 molecular function GO: 0003735 structural constituent of ribosome 11 5.00E-07 GO: 0035662 Toll-like receptor 4 binding 3 5.10E-06 cellular component GO: 0044445 cytosolic part 12 1.20E-06 GO: 0022626 cytosolic ribosome 9 1.60E-06 GO: 0022625 cytosolic large ribosomal subunit 7 4.10E-06 GO: 0044391 ribosomal subunit 10 9.50E-06 GO: 0005840 ribosome 11 1.30E-05 GO: 0005683 U7 snRNP 3 2.90E-05 GO: 0005687 U4 snRNP 3 8.40E-05 GO: 0005615 extracellular space 29 0.00012 GO: 0015934 large ribosomal subunit 7 0.00014 GO: 0005576 extracellular region 58 0.00016 GO: 0032991 macromolecular complex 61 0.00016 GO: 0034709 methylosome 3 0.00018 GO: 0044421 extracellular region part 52 0.00023 GO: 0034719 SMN-Sm protein complex 3 0.00033  DOWN-REGULATED  cellular component GO: 0044424 intracellular part 839 3.60E-30 GO: 0005622 intracellular 858 1.50E-27 GO: 0043229 intracellular organelle 737 6.00E-22 GO: 0043226 organelle 787 1.90E-21 GO: 0005737 cytoplasm 660 1.30E-19 GO: 0043227 membrane-bounded organelle 724 7.80E-18 GO: 0044446 intracellular organelle part 511 1.30E-15 GO: 0043231 intracellular membrane-bounded organelle 630 2.10E-15 GO: 0044422 organelle part 519 5.80E-15 molecular function GO: 0005515 protein binding 567 9.00E-22 biological process GO: 0044763 single-organism cellular process 641 2.60E-18 GO: 0007275 multicellular organism development 364 2.70E-17 GO: 0048731 system development 333 5.90E-17 GO: 0009888 tissue development 166 2.00E-16 GO: 0048518 positive regulation of biological process 379 2.50E-15 GO: 0048513 animal organ development 260 3.70E-15 GO: 0048856 anatomical structure development 383 3.90E-15 GO: 0043588 skin development 48 4.90E-15 GO: 0044707 single-multicellular organism process 407 5.00E-15 GO: 0032502 developmental process 407 1.10E-14Figure 7
Genes were clustered in the PPI network according to string database (genes from top 10 predicted pathways in BQG-treated group versus BLM group).


### 3.6. Notch-1 and Jagged-2 Play The Important Roles in The 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 Figures8(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.Figure 8
Validation of RT-qPCR. The predicted target genes, such as Cxcl2 (a), Snap25 (b), Eif3j2 (c), Notch-1 (d), and Jagged-2 (e), were selected for validation by RT-qPCR. Data were expressed as fold of changes compared to the control group from at least three biological replicates.
(a) (b) (c) (d) (e)Figure 9
Validation of Western blotting. (a) Representative proteins of Notch signaling were detected exposed to BLM with or without BQG treatment in mice skin tissues. (b) Relative protein expression of Notch-1/GAPDH. (c) Relative protein expression of Jagged-2/GAPDH.∗P<0.05, ∗∗P<0.01 compared with BLM model group.
(a) (b) (c)


## 3.1. Typical Chemical Components and Their 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 Figures1(a) and 1(b) and Table 3, a total of 14 peak signals and the content of each constituent were identified and calculated, respectively.Table 3
Identification of typical chemical components and its contents in BQG.
Peak Identification Content (ug/g) Source 1 Amygdalin 2023.5 TaoRen 2 Paeonol 764.5 DanPi 3 Schisandrol A 38.7 WuWeiZi 4 Dihydrotanshinone I 9.3 DanShen 5 Cryptotanshinone 11.5 DanShen 6 Schizandrin A 0.7 WuWeiZi 7 Tanshinone IIA 9.9 DanShen 8 Schizandrin B 0.7 WuWeiZi 9 Danshensu 2290.2 DanShen 10 Acteoside 549.3 LingXiaoHua 11 Ferulic acid 45.2 DangGui 12 Lobetyolin 31.0 DangShen 13 Platycodin D 76.2 JieGeng 14 Astragaloside IV 196.0 HangQiFigure 1
Fingerprints of BQG samples in the positive (a) and negative (b) ion modes by UHPLC-Q-TOF/MS. Peak 1, Amygdalin; Peak 2, Paeonol; Peak 3, Schizandrol A; Peak 4, Dihydrotanshinone I; Peak 5, Cryptotanshinone; Peak 6, Schizandrin A; Peak 7, Tanshinone IIA; Peak 8, Schizandrin B; Peak 9, Danshensu; Peak 10, Acteoside; Peak 11, Ferulic acid; Peak 12, Lobetyolin; Peak 13, Platycodin D; Peak 14, Astragaloside IV.
(a) (b)


## 3.2. 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 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).Figure 2
Overall health assessment after BQG treatment in SSc mouse model. (a) The curves of body weight of mice in different groups at different time points. (b) The percentage of lung, spleen, and thymus index, n=6 per group;∗P<0.05, ∗∗P<0.01 compared with the model group.
(a) (b)


## 3.3. 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 BLM-induced 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 (Figures3(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)).Figure 3
Effects of BQG in improving BLM-induced skin sclerosis of mouse model. Skin sections derived from BLM or PBS-treated mice in the presence or absence of BQG administration were stained with hematoxylin and eosin (a) and Masson’s trichrome (b). Representative images are shown. Scale bar=100μm. (c) Data of dermal thickness are presented by histogram. (d) The levels of HYP content in each group of mice.In addition,α-smooth muscle actin (α-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.Figure 4
α-SMA expression was evaluated by immunohistochemistry. Black arrows indicated α-SMA-positive cells in vascular walls. Representative images are shown. Original magnifications are 200× (scale bar=100 μm) and 400× (scale bar=50 μm), respectively.Figure 5
ELISA assay showed elevated Cxcl2 in the serum derived from mice in different groups. Each graph illustrates mean ± SD of the indicated parameters (n≥3 per group;∗P<0.05, ∗∗P<0.01 vs 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.


## 3.4. 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 Figure6(a), both up- and downregulated DEGs were overlapped. Of these, transcriptomic analysis revealed that, 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).Table 4
Upregulated DEGs after intervention of BQG.
Gene ID Gene Name log2 fold change P-value ENSMUSG00000097656 Gm26712 7.98659314 1.31E-06 ENSMUSG00000105338 Gm43802 7.80359975 3.28E-08 ENSMUSG00000106587 AC125099.2 7.470051892 3.57E-06 ENSMUSG00000103308 Gm37800 4.023628692 4.11E-05 ENSMUSG00000049421 Zfp260 3.689570886 9.22E-06 ENSMUSG00000003545 Fosb 3.68790389 3.35E-06 ENSMUSG00000057465 Saa2 3.572774437 2.45E-06 ENSMUSG00000079025 Gsdmc 3.355515254 8.02E-06 ENSMUSG00000043424 Eif3j2 3.102051328 2.66E-07 ENSMUSG00000046311 Zfp62 2.135549165 1.47E-06Table 5
Downregulated DEGs after intervention of BQG.
Gene ID Gene Name log2 fold change P-value ENSMUSG00000027273 Snap25 -6.26729 1.36E-05 ENSMUSG00000084257 Gm11597 -6.26702 8.33E-05 ENSMUSG00000083405 Gm15725 -5.90441 6.95E-07 ENSMUSG00000102564 Gm37035 -5.4123 6.22E-08 ENSMUSG00000058427 Cxcl2 -5.30387 4.51E-06 ENSMUSG00000097724 Gm26850 -5.06965 1.66E-05 ENSMUSG00000099068 Gm27861 -4.83004 4.91E-05 ENSMUSG00000029379 Cxcl3 -4.54472 8.15E-05 ENSMUSG00000097078 Gm26566 -4.25859 9.63E-05 ENSMUSG00000103309 BC037039 -4.22422 3.50E-05Figure 6
Transcriptomic assay and bioinformatics analysis. (a) Venn diagram of overlapping genes derived from transcriptome analysis in a pairwise comparison. (b) Compared with BLM group, filtering of significant DEGs was presented by valcano. (c) Heat map of regulated DEGs in skin samples of control, BLM, and BQG-treated groups. Red color represents upregulated genes, while green color shows downregulated genes. The depth of color represents difference on multiplier.
(a) (b) (c)


## 3.5. 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 Table6. Notch signaling pathway ranking 5 was priorly chosen to be 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 of cellular component, which had the enriched DEG called Notch-1. Both up- and downregulated mRNAs significantly enriched GO terms, and the top 20 DEGs are presented in Table 7.Table 6
Signaling pathway prediction of DEGs between BQG and BLM group from KEGG database.
ID Description P-value Significant symbol genes UP-REGULATED  ko03010 Ribosome 0.001525 Rpl30, Rpl26, Rpl35a, Rpl31, Rps27a, Rpl27-ps3 ko04924 Renin secretion 0.002328 Calm4, Gucy1a3, Adcyap1r1, Gucy1b3 ko03030 DNA replication 0.004173 Rnaseh2b, Rpa3, Mcm6 ko04624 Toll and Imd signaling pathway 0.004522 Birc3, Tab2, Faf1 ko04713 Circadian entrainment 0.008117 Calm4, Gucy1a3, Adcyap1r1, Gucy1b3 ko05031 Amphetamine addiction 0.018481 Calm4, Fosb, Ppp1cb ko00230 Purine metabolism 0.018499 Nt5e, Entpd5, Gucy1a3, Gucy1b3, Pde4d ko04970 Salivary secretion 0.020157 Calm4, Gucy1a3, Gucy1b3 ko04611 Platelet activation 0.022505 Gucy1a3, Vamp8, Ppp1cb, Gucy1b3 ko04270 Vascular smooth muscle contraction 0.023148 Calm4, Gucy1a3, Ppp1cb, Gucy1b3  DOWN-REGULATED  ko04550 Signaling pathways regulating pluripotency of stem cells 0.001454 Inhbb, Wnt4, Map2k2, Dvl1, Mapk13, Id1, Fgfr3, Axin2, Tcf3, Myf5, Wnt9a, Fzd8, Fzd2, Dlx5, Wnt3a, Fgfr4 ko04360 Axon guidance 0.002752 Wnt4, Sema3f, Sema4g, Efnb1, Plxna3, Ssh3, Epha2, Pak6, Abl1, Pak4, Ephb3, Plxnb1, Efna2, Fes, Nck2, Ephb6, Sema4f, Sema4b ko04115 p53 signaling pathway 0.002898 Sfn, Gtse1, Gadd45g, Gadd45a, Rprm, Gadd45b, Pidd1, Sesn2, Bbc3, Bax ko04390 Hippo signaling pathway 0.003971 Wnt4, Dvl1, Llgl2, Id1, Axin2, Llgl1, Wnt9a, Fzd8, Fzd2, Wwc1, Scrib, Tead3, Ajuba, Wnt3a, Bbc3, Smad7 ko04330 Notch signaling pathway 0.006239 Dvl1, Hes1, Dtx2, Lfng, Jag2, Notch1, Ncor2 ko05166 HTLV-I infection 0.006668 Wnt4, Dvl1, Cdc20, Ranbp3, Ranbp1, Crtc1, Map3k14, Tcf3, Pold1, H2-Q2, Wnt9a, Msx1, Bcl2l1, Fzd8, Fzd2, Cdkn2b, H2-Q6, Ltbr, Wnt3a, Pdgfa, Slc2a1, Mybl2, Bax, Fosl1 ko04014 Ras signaling pathway 0.010254 Ngfr, Map2k2, Pla2g6, Rgl2, Arf6, Gnb2, Fgfr3, Rin1, Epha2, Pak6, Abl1, Pak4, Rasal3, Pla2g4f, Efna2, Bcl2l1, Zap70, Pdgfa, Fgfr4, Rasal1 ko05322 Systemic lupus erythematosus 0.012114 Hist1h2bl, Hist3h2a, Hist1h2br, Hist1h4n, Hist1h4m, H2afz, Hist1h3d, H2afj, Hist1h4k, Hist2h3c2, Hist1h3i, Hist1h2bg, Hist1h2bk, H2afx ko00680 Methane metabolism 0.012152 Shmt2, Eno1b, Pfkl, Phgdh, Eno1 ko05230 Central carbon metabolism in cancer 0.014355 Slc16a3, Map2k2, Fgfr3, Hk3, Pfkl, Sirt6, Erbb2, Slc2a1Table 7
The significantly enriched GO terms of DEGs ranking top 20.
Ontology ID Term Gene count P-value UP-REGULATED  biological process GO: 0008152 metabolic process 119 4.60E-07 GO: 0044237 cellular metabolic process 107 4.20E-06 GO: 0044238 primary metabolic process 108 6.30E-06 GO: 0071704 organic substance metabolic process 111 6.90E-06 molecular function GO: 0003735 structural constituent of ribosome 11 5.00E-07 GO: 0035662 Toll-like receptor 4 binding 3 5.10E-06 cellular component GO: 0044445 cytosolic part 12 1.20E-06 GO: 0022626 cytosolic ribosome 9 1.60E-06 GO: 0022625 cytosolic large ribosomal subunit 7 4.10E-06 GO: 0044391 ribosomal subunit 10 9.50E-06 GO: 0005840 ribosome 11 1.30E-05 GO: 0005683 U7 snRNP 3 2.90E-05 GO: 0005687 U4 snRNP 3 8.40E-05 GO: 0005615 extracellular space 29 0.00012 GO: 0015934 large ribosomal subunit 7 0.00014 GO: 0005576 extracellular region 58 0.00016 GO: 0032991 macromolecular complex 61 0.00016 GO: 0034709 methylosome 3 0.00018 GO: 0044421 extracellular region part 52 0.00023 GO: 0034719 SMN-Sm protein complex 3 0.00033  DOWN-REGULATED  cellular component GO: 0044424 intracellular part 839 3.60E-30 GO: 0005622 intracellular 858 1.50E-27 GO: 0043229 intracellular organelle 737 6.00E-22 GO: 0043226 organelle 787 1.90E-21 GO: 0005737 cytoplasm 660 1.30E-19 GO: 0043227 membrane-bounded organelle 724 7.80E-18 GO: 0044446 intracellular organelle part 511 1.30E-15 GO: 0043231 intracellular membrane-bounded organelle 630 2.10E-15 GO: 0044422 organelle part 519 5.80E-15 molecular function GO: 0005515 protein binding 567 9.00E-22 biological process GO: 0044763 single-organism cellular process 641 2.60E-18 GO: 0007275 multicellular organism development 364 2.70E-17 GO: 0048731 system development 333 5.90E-17 GO: 0009888 tissue development 166 2.00E-16 GO: 0048518 positive regulation of biological process 379 2.50E-15 GO: 0048513 animal organ development 260 3.70E-15 GO: 0048856 anatomical structure development 383 3.90E-15 GO: 0043588 skin development 48 4.90E-15 GO: 0044707 single-multicellular organism process 407 5.00E-15 GO: 0032502 developmental process 407 1.10E-14Figure 7
Genes were clustered in the PPI network according to string database (genes from top 10 predicted pathways in BQG-treated group versus BLM group).


## 3.6. Notch-1 and Jagged-2 Play The Important Roles in The 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 Figures8(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.Figure 8
Validation of RT-qPCR. The predicted target genes, such as Cxcl2 (a), Snap25 (b), Eif3j2 (c), Notch-1 (d), and Jagged-2 (e), were selected for validation by RT-qPCR. Data were expressed as fold of changes compared to the control group from at least three biological replicates.
(a) (b) (c) (d) (e)Figure 9
Validation of Western blotting. (a) Representative proteins of Notch signaling were detected exposed to BLM with or without BQG treatment in mice skin tissues. (b) Relative protein expression of Notch-1/GAPDH. (c) Relative protein expression of Jagged-2/GAPDH.∗P<0.05, ∗∗P<0.01 compared with BLM model group.
(a) (b) (c)


## 4. 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 fromSalvia 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 ofAstragalus 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 α-SMA plays an important role in the pathogenesis of SSc [31, 32]. BQG was showed to be advantageous to improve the expression level of α-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 γ-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 [38–40]. 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.Figure 10
Schematic illustration of potential signaling pathways. Schematic diagram of BQG potential protective effects against skin fibrosis by regulating targets of Notch signaling pathway.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.


## 5. 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.



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*Source: 6709278-2019-07-02.xml*