Label-Free Quantification based Proteomic Analysis of Serum Obtained from Henoch– Schönlein Purpura Patients before and after Zhenbao Pill Treatment

Henoch–Schönlein Purpura (HSP), also known as immunoglobulin A vasculitis, is an immune disorder that is predominantly seen in children (8–20/100000 each year) worldwide. It is characterized by inflammation and bleeding of tiny blood vessels in the skin, joints, intestines, and kidneys. The most distinctive characteristic of this type of vasculitis is a purple rash that often affects the thighs and buttocks. The Zhenbao pill, containing several medicinally important compounds, has been found to be effective against a variety of neurological and immunological illnesses. We used a label-free quantification (LFQ) proteomics approach to investigate the effect of the Zhenbao pill on the differential serum protein expression of HSP patients. Out of 14 significantly differentially expressed proteins, four proteins immunoglobulin lambda variable 3-27, immunoglobulin kappa variable 3-11, neural cell adhesion molecule L1-like protein, and immunoglobulin heavy variable 3-33 were upregulated, whereas 10 proteins i.e., protein Z-dependent protease inhibitor, C4b-binding protein alpha chain, heparin cofactor 2, complement C4-A, complement C4 beta chain, vitamin K-dependent protein Z, coagulation factor VII, proteoglycan 4, lipopolysaccharide-binding protein (LBP), thrombospondin-4, and plasminogen activator inhibitor 1 were downregulated. Bioinformatics analysis of these differentially expressed proteins revealed that they play a critical role in immune-related responses. Parallel reaction monitoring validation also substantiated the results of the LFQ proteomics approach by analyzing the expression of LBP and SERPINA10. This work identifies the proteins and protein-related molecular pathways that are responsible for the efficacy of Zhenbao pill in the management of HSP.


INTRODUCTION
H enoch-Schönlein purpura (HSP) is commonly found in children, with a prevalence of 8-20 children per 100,000 each year. Its incidence is higher in males than in females (1.5:1) and children between the ages of 3 and 8 years are particularly vulnerable (Di Pietro et al., 2019;Leung et al., 2020;Rostoker, 2001). HSP is a systemic small-vessel vasculitis caused by immunoglobulin A (IgA) accumulation in the vessel walls. This results in damage to O n l i n e
Various therapeutic approaches have been used to treat HSP; however, none have yet proven effective. The Zhenbao pill consists of 29 Chinese herbal medicines, including pearl, bezoar, amomum, saffron, cassia seed, and licorice, which promotes blood circulation, calms nerves, and activates meridians (Jia et al., 2021). It has been reported to be anti-inflammatory drug effective against various disorders (Luo et al., 2020). The Zhenbao pill has been used against certain neurological disorders, including hemiplegia sequelae and stroke. Studies have revealed that the Zhenbao pill leads to the eradication of oxygen free radicals, promotes microcirculation and repairs damaged neurons (He et al., 2018a;Liu et al., 2015;Wu et al., 2007). One study found that the Zhenbao pill had a protective effect on the nerves of rats; the Basso-Beattie-Bresnahan score of rats with spinal cord injury (SCI) was improved after Zhenbao pill administration, and a reduction in the proportion of Treg cells was also observed (Bluman and Goldman, 2014;Rostoker, 2001). It has recently been established that SCI repair can be promoted directly by reducing Treg cell numbers and indirectly by alleviating immunosuppression (Raposo et al., 2014). The mechanism behind this is related to the inhibition of Treg cell numbers, which leads to the reduction of transforming growth factor-β levels by inhibiting the miR-214-induced heat shock protein 27 expression during SCI repair (He, Yongxiong et al., 2018a). The above literature illustrates that Zhenbao pill has been found effective against various immune related disorders. However, further research is required to elucidate the underlying molecular mechanism of action of the Zhenbao pill in these immune disorders.
A label-free quantification (LFQ)-based proteomics (Al-Shweiki et al., 2017) investigation was conducted to understand the effect of the Zhenbao pill on protein-based molecular pathways in the serum of HSP patients before and after its administration. This investigation will aid in understanding the molecular processes that are responsible for its effectiveness against immunological dysfunctions and evaluating its potential as an effective therapeutic strategy against HSP.

Patients and serum samples
The research protocol was approved by the Ethics Committee of the Affiliated Hospital of Inner Mongolia University for the Nationalities (NM-LL-2019-01-05-01). HSP patients were diagnosed in the Affiliated Hospital of Inner Mongolia University for the Nationalities. Diagnostic criteria of HSP were in accordance with the guidelines for HSP diagnosis and treatment, which include: skin purpura, joint swelling and pain, abdominal pain and hematochezia. The main manifestations of renal involvement are hematuria and proteinuria.
Inclusion other complications including infection, cancer, hypertension, diabetes and other systemic diseases to meet the criteria. Written informed consent was obtained from all the participants. The detailed information pertaining to the HSP patients, including their demographics, and main manifestation are provided in Table I. HSP patients admitted to this study were provided daily with 3-5 g of Zhenbao pill (based on the body weight). The effect of HSP treatment with Zhenbao pill was followed every two weeks up to four weeks by examining the urine albumen and blood.
In this study, 30 blood samples from HSP patients were collected. Among them, 12 blood samples were randomly selected for proteomics. Before centrifugation O n l i n e

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at 4°C (1000×g), the samples were allowed to coagulate at room temperature to obtain the serum. All serum samples were tested at the same time, and they were stored at -80 °C in the freezer until use. Whole of the experimental scheme is shown in Figure 1. Table include characteristics, Before Zhenbao pill treatment, after Zhenbao pill treatment. Fig. 1. Flow chart of the experimental scheme. HSP patients were treated with Zhenbao pill, samples were collected and processed. A label-free quantification proteomics was performed and data was analyzed with bioinformatics tools. Proteins were validated by PRM.

Serum treatment and protein digestion
The serum samples were thawed and depleted of albumin, IgG, and haptoglobin by using the albumin/ IgG SpinTrap columns (GE Healthcare) according to the manufacturer's protocol. Total protein concentration was determined using the Bradford protein assay (Quick Start Bradford protein assay kit; Bio-Rad, USA), and 15 μg of protein from each sample was loaded and separated using 12% SDS-PAGE gels, which were subsequently stained with the Coomassie blue reagent (Bio-Rad). Gel bands were then cut into 1-2-mm 3 cubes, washed twice with destain buffer [500 μL, 50 mM NH 4 HCO 3 in acetonitrile (ACN,1:1), v/v] for 20 min, dehydrated with ACN (500 μL), and dried in a SpeedVac (ThermoFisher). The proteins were reduced in 10 mM dithiothreitol (50 μL) at 56 °C for 30 min. Alkylation was completed using 55 mM iodoacetamide (50 μL) for 30 min in the dark at room temperature. Gel bands were shrunk with ACN (500 μL) for 15 min. The gel bands were completely dried before trypsin digestion and rehydrated using 0.01 μg/μL trypsin (15-20 μL). The proteins were then incubated at 37 °C overnight. The gel fragments were heated in a 40 °C water bath for 60 min and sonicated for 3 min every 30 min.
Subsequently, the supernatant was transferred to clean tubes, and the gel fragments were extracted with 200 μL 50% ACN/2.5% trifluoroacetic acid (v/v) at 30 °C for 60 min and sonicated for 3 min every 30 min. The peptides were dried in a SpeedVac and re-solubilized in 0.1% formic acid (FA).

LC-MS/MS analysis
The peptides were separated using a nano-liquid chromatography system (EASY-nLC 1200, Thermo Fisher Scientific) in conjunction with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific). Trapped samples were separated on an Acclaim PepMap C18 trap column (100 mm×2 cm, 5 mm) after separation on an Acclaim PepMap C18 separation column (100 mm×50 cm, 2 mm). The samples were pumped through the columns at a flow rate of 600 nL/min. Mobile phase A included 1.9 % ACN and 0.1 % FA in H 2 O(v/v), whereas mobile phase B contained 1.9 % H 2 O and 0.1 % FA in ACN (v/v). The gradient began at 6% B, increased to 9% B in 8 min, and then to 9-14 % buffer B for 8-24 min, 14-30 % buffer B for 24-60 min, 30-40 % buffer B for 60-75 min, 40-95 % buffer B for 75-78 min, and 95 % buffer B for 78-85 min, before returning to 6% B within 1 min and equilibrating for 4 min.
MS spectra were collected using the following parameters: automated gain control (AGC) target, 1e6; injection duration, 50 ms; mass range, 400-1500 mass to charge ratio (m/z); and resolution, 35,000. The MS/MS scans were performed on the top ten precursor ions using a normalized collision energy of 30 % and a dynamic exclusion time of 30 s. MS spectra were obtained in the positive mode with a resolution of 17,500, an AGC target of 2e5, and an injection period of 50 ms.

Protein identification and LFQ
MaxQuant version 1.5.2.8 was applied to evaluate the raw data, which were compared to the Uniprot Human reference database (20,377 protein entries, version of June 2020). The search parameters included a 20-ppm mass tolerance for precursors, a 20-ppm mass tolerance for fragments, trypsin cleavage, and up to two miscleavages. The minimum peptide length was set to seven amino acid residues, and carbamidomethylation of cysteine residues was treated as a fixed modification, whereas oxidation (M) and acetylation (protein N-terminal) were treated as variable modifications. At both the peptide and protein levels, the incidence of false discovery was less than 1%. LFQ and intensity-based absolute protein quantification (iBAQ) were utilized. The two groups were compared using a log2 transformation of the iBAQ results and a twosample t-test. Differentially expressed proteins (DEPs)

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must fulfill the following criteria: fold change more than 1.5 or equal to 0.67 and a p value less than 0.05.

Bioinformatics analysis of DEPs
Analysis of DEPs was carried out by using OmicsBean (http://www.omicsbean.cn/) (Iqbal et al., 2018(Iqbal et al., , 2019, a multi-omics data analysis tool that includes Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO) enrichment, and protein-protein interaction (PPI) network analyses.

Parallel reaction monitoring (PRM) validation
Only DEPs identified with a high confidence peptide sequence were selected for parallel reaction monitoring (PRM) validation based on proteomics data. Three biological replicates were included, each of which was performed with 1-μg samples. First, trypsin-digested peptides were dissolved in buffer A (1.9 % ACN and 0.1 % FA in H 2 O,v/v ) and eluted using an Acclaim PepMap C18 (75 μm × 25 cm) with gradient buffer B (1.9 % H 2 O and 0.1 % FA in CAN, v/v; 6% to 9% B in 8 min, 9% to 14% B over 16 min, 14% to 30% B for 36 min, 30% to 40% B for 15 min, 40% to 95% B for 5 min, holding at 95% B for 7 min, decreased to 6% B in 1 min, and kept at 5% for the last 4 min) at a flow rate of 300 nl/min. The eluted peptides were analyzed with a nano spray ionization source and Q-Exactive HFx (Thermo Fisher Scientific). MS1 spectra were measured in the orbitrap at 35,000 resolutions, with a mass range from 400 to 1500 m/z, maximum injection time of 30 ms, and dynamic exclusion of 45 s. The data-dependent mode cycle was set to trigger MS/MS on up to the top 20 most abundant precursors per cycle at an MS 2 resolution of 17,500, AGC target of 2e5, maximum injection time of 50 ms, isolation window of 1.6 m/z, and HCD collision energy of 27. Precursor ions with charge states z = 2-6 was selected for MS/MS collisioninduced dissociation fragmentation. The PRM data were processed using the Skyline software (version 20.2.0.286, MacCoss Lab, University of Washington, United States) (Adams et al., 2020). The results for each peptide were quantified according to the MS/MS ion peak area from its corresponding transitions, and statistical significance was set at p < 0.05 using the Student's t-test.

RESULTS
LFQ-based proteomics approach produced interesting results. A total of 389 DEPs were identified, of which 14 proteins were significantly differentially expressed (p value < 0.05; Fig. 2B). Four proteins immunoglobulin lambda variable 3-27, immunoglobulin kappa variable 3-11, neural cell adhesion molecule L1-like protein, immunoglobulin heavy variable 3-33 were upregulated, whereas 10 proteins protein Z-dependent protease inhibitor, C4b-binding protein alpha chain (C4BP), heparin cofactor 2, complement C4-A, vitamin K-dependent protein Z, coagulation factor VII, proteoglycan 4, lipopolysaccharide-binding protein (LBP), thrombospondin-4, and plasminogen activator inhibitor 1 were downregulated (Table II).  Bioinformatics analysis was performed by OmicsBean bioinformatics tool to retrieve complete biological information of these DEPs. A summary of the total GO hits is shown in Figure 2A, which illustrates the total quantitative biological processes (BP), cellular components (CC), molecular functions (MF), and KEGG pathways. Fifteen enriched BP are shown in Figure 3, illustrating the involvement of these DEPs in immunerelated dysfunctions after Zhenbao pill treatment. These processes include response to external stimulus, response to stress, positive regulation of the immune system process, leukocyte migration, response to wounding, regulation of response to external stimulus, regulation of response to wounding, protein maturation, vesicle-mediated transport, regulation of protein metabolic process, import into cell, protein processing, regulation of protein maturation, regulation of inflammatory response and regulation of protein processing. The involvement of these DEPs in the CC of the Zhenbao pill-treated cells is shown in Figure 4A. Most of the enriched proteins were found to be involved in extracellular components, whereas few were involved in immunologic responses. KEGG analysis (Fig. 4B) revealed involvement of the complement and coagulation cascade, which directly involves immunityrelated functions. Figure 5 shows the PPI network of DEPs. Several biological pathways related to these DEPs interact with each other. These include the complement and coagulation cascades, NF-kappa B signaling pathway, p53 signaling pathway, toll-like receptor signaling pathway, and ECM signaling pathway.

PRM validation
The expression of 14 DEPs was underwent PRM analysis to further validate the mass spectrometric results. Owing to the requirements of protein characteristics and abundance, we obtained abundant values of six proteins using quantitative data of target peptide fragments, including C4BPA, SERPIND1, F7, C4A, LBP, and SERPINA10. Among them, SERPINA10 and LBP successfully validated the proteomics results. As shown in Figure 6, these proteins showed exactly the same trend as when quantified by using the LFQ proteomics method, although the fold change values varied between the two techniques. Significant differences were observed between the groups (Supplementary Tables S1-S3).

DISCUSSION
This study investigated the impact of the Zhenbao pill on protein expression in the serum of patients with HSP. Although the mechanism of HSP remains unclear, various hypotheses have been proposed to highlight the pathophysiology of HSP. There are many different types of immunoglobulin A nephritis (IgAN), immunoglobulin A vasculitis (IgAV), and immunoglobulin. Vasculitis nephropathy (IgAVN) may develop in people. It is possible that all forms contain the same entities. IgAV, IgAVN, and IgAN may have different origins, depending on the type of tissue in which they are detected. Immune complexes in IgAN may be formed due to increased galactose-deficient O n l i n e

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IgA1 levels (Gd-IgA1), which can be explained by a combination of hereditary and environmental variables. IgA1 is responsible for the IgAN recognition of the endothelial cell antigen (Heineke et al., 2017;Knoppova et al., 2016;Novak et al., 2015).  Gd-IgA1 can bind to immune complexes, such as CD89 and CD71 and to complement components that could be receptors of IgA. Human mesangium cells are activated through the deposition of Gd-IgA1-containing immune complexes, resulting in abnormal renal function (Heineke et al., 2017;Knoppova et al., 2016;Novak et al., 2015). IgAV and IgAVN seem to have overlapping pathological mechanisms. The interplay between IgA and FcRI receptors may activate and attract neutrophils to areas of inflammation and is subject to changes due to environmental factors or through specific molecular pathways (Yeo et al., 2018). This leads to an increase in the levels of IgA1 AECA in blood; IgA1 AECA then bind to tiny vessels. The interaction of AECA with endothelial cells results in the production of interleukin-8, a neutrophil chemoattractant (Lau et al., 2010;Yeo et al., 2018).
The interaction between IgA1 and FcRI stimulates neutrophil recruitment. Processes, such as CDC, ADCC, NETosis, and ROS generation, harm vascular endothelial cells. LTB4 is generated by IgA-activated neutrophils, which activates and attracts additional neutrophils. Furthermore, neutrophils release TNF, which stimulates endothelial cells and reveals antigens that are normally buried. After identification by AECA antibodies, these antigens may eventually bind to the endothelium antigens. Neutrophil activation eventually results in inflammation and vascular hemorrhage, as seen in IgAV and IgAVN (Heineke et al., 2017;Yang et al., 2002Yang et al., , 2004Yang et al., , 2006Yang et al., , 2012. HSP is an autoimmune condition triggered by pathogens, environmental factors, or hereditary factors (Yeo et al., 2018). Specific immune system abnormalities are responsible for HSP pathogenesis. As seen in Figure 2A, the Zhenbao pill dysregulated proteins that further influenced immune-related biological processes, demonstrating that the Zhenbao pill may have a therapeutic effect on HSP. Zhenbao pill is made up of almost 29 Chinese herbal medicines, including cassia seed, pearl, amomum, saffron, licorice, and bezoar, which help in activating meridians, promoting blood circulation, and calming nerves. The Zhenbao pill can promote microcirculation, repair damaged neurons, and remove oxygen free radicals (Kubo et al., 2007;Liu et al., 2015;Wu et al., 2007).
LFQ proteomics analysis revealed the upregulation of 3 immunoglobulin proteins: Immunoglobulin kappa variable 3-11, immunoglobulin lambda variable 3-27 and immunoglobulin heavy variable 3-3, after treatment with the Zhenbao pill. The immune system releases immunoglobulins or Y-shaped proteins to identify and phagocytose foreign bodies, such as viruses, bacteria, and other pathogens. Such infection is responsible for the production of unique antigen molecules that are identified by antibodies (Aleyd et al., 2015;He, Yongxiong et al., 2018a, b;He et al., 2018;Liu et al., 2015;Nicholson, 2016;Wu et al., 2007). Paratopes are present in antibodies; each antigen has a specific epitope that mimics a lock and key mechanism, enabling the two structures to be linked with the accuracy of the pinpoint. Paratoperase is found at the tip of the antinodes (Y) (Sela-Culang et al., 2013). An antibody is either an infected cell or a microbe that is assaulted by other components in the immune system, or directly neutralized by the binding mechanism by blocking the virus part that is critical for invasion (Aleyd et al., 2015).
Antibodies are a component of the adaptive immune system, along with B and T cells. A B cell-attached form and a soluble form, both of which may be detected in extracellular fluids, such as blood plasma. Initially, all antibodies are in the form of B cell receptors (BCR), which are connected to the surface of a BCR. To produce soluble antibodies with a similar paratope to those secreted by the plasma cells, the antigens bind with the BCR to remain in the body for a long time and produce immunity against the O n l i n e

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Label-Free Quantification based Proteomic Analysis of Serum Obtained 7 antigen (Janeway, 2001). Additionally, soluble antibodies can be released into the tissue fluid, blood stream, and other secretions. These fluids are historically referred to as humor and are responsible for exhibiting humoral immunity or antibody-mediated immunity (Litman et al., 1993). Upregulation of these immunoglobulins may have a protective role during HSP infections when treated with Zhenbao pill. Neural cell adhesion molecule L1-like protein (CHL1) is a multidomain type 1 membrane glycoprotein, belonging to the immunoglobulin superfamily, whose expression is perturbed when HSP patients are treated with the Zhenbao pill. It performs similar physiological activities in neural system development as CHL1 (Borghesi and Milcarek, 2006). CHL1 is produced as two isoforms, and isoform two is distinguished by the presence of an extra miniexon 8 (Karstens et al., 2020). These isoforms have no physiological significance. CHL1 is involved in nerve cell regeneration and the growth, proliferation, and migration of neurons and serves to preserve motoneurons (Holm et al., 1996;Pier, 2004) (Guseva et al., 2018;Holm et al., 1996;Karstens et al., 2020). The increased CHL1 levels in patients with HSP after treatment with the Zhenbao pill illustrates the recovery processes of vessels and skin tissues.
Hepatocyte-derived C4BP is another protein with differential expression that serves as an essential component of the classic complement pathway. Here, it serves as a cofactor for serine protease factor I (F) in the degradation of C4b (Guseva et al., 2018;Jakovcevski et al., 2009). It inhibits the formation of the C4b2a complex (C3 convertase) that plays an important role in the inflammatory process following the activation of the signaling pathway. In human plasma, there are many distinct C4BP owing to the combinations of its C4BPα chain (75 kDa) and C4BPβ chain (Jakovcevski et al., 2009). Six or seven chains (C4BP) and either one or no chains (C4BP) are present in C4BP, depending on the model (Jakovcevski et al., 2009). In C4b, the anticoagulant protein S (PS) is bound by C4BP, whereas C4BP binds to the same protein. In addition to be a cofactor of activated protein C (APC), PS is a vitamin K-dependent plasma glycoprotein (Mr 75 000) (APC). Blood coagulation factors, FVa and FVIIIa, are inactivated by APC (Dahlbäck, 1991;Rezende et al., 2004;Vehar and Davie, 1980). Therefore, PS may also directly block the prothrombinase complex by binding to FVa and FXa (Vehar and Davie, 1980;Walker, 1981).
Heparin cofactor II is another dysregulated protein found in patients with HSP treated with the Zhenbao pill that is involved in various immune functions. It is associated with leukocyte-mediated protein breakdown, which releases cytokines as the inflammatory response (Heeb et al., 1994). Although it is a thrombin inhibitor, the absence of HCII does not result in significantly higher levels of thrombosis (Heeb et al., 1993(Heeb et al., , 1994. Levels of PAI-1 were downregulated in this study. This protein inhibits the activity of urokinase plasminogen activator (uPA) that plays a role in plasmin formation by cleaving palminogen (Mahmood et al., 2018). Plasmin accelerates the breakdown of the extracellular matrix directly or indirectly with the help of matrix metalloproteinases. In this scenario, PAI-1 inhibits uPA via active site binding, preventing plasmin formation. Additional inhibition is mediated by PAI-1 binding to the uPA/uPA receptor complex, resulting in the degradation of the latter (Heeb et al., 1994;McMahon and Kwaan, 2015). Thus, PAI inhibit the serine proteases tPA and uPA/ urokinase and consequently fibrinolysis, the physiological process that degrades blood clots. PAI-1 inhibits the activity of matrix metalloproteinases, which play a crucial role in the invasion of malignant cells through the basal lamina. PAI-1 is mainly produced by the endothelium (cells lining blood vessels) but is also secreted by other tissues, such as adipose tissue (Qureshi et al., 2016).

CONCLUSION
The present study is an attempt to explore some sound therapeutic strategy against HSP. Therefore, Zhenbao pill has been used in HSP patients and their proteomic profile is checked using LFQ proteomics approach. Expression of various proteins of vital importance is found to be perturbed in HSP patients when treated with Zhenbao pill. For example, proteins involved in positive regulation of the immune system, leukocyte migration, response to wounding, vesicle mediated transport, regulation of protein metabolic process, protein activation cascade, protein processing, regulation of protein maturation, regulation of inflammatory response, acute inflammatory response, regulation of protein processing, and regulation of complement activation are immune related cascades affected when treated with Zhenbao pill. In summary, Zhenbao pill may prove to be effective in certain immunerelated functions and may be beneficial as a new therapeutic agent against HSP in future.

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Wuritunashun et al.

Supplementary material
There is supplementary material associated with this article. Access the material online at: https://dx.doi. org/10.17582/journal.pjz/20220623100642

Statement of conflict of interest
The authors have declared no conflict of interest.