A Combination of UPLC-LTQ-Orbitrap-MSn Research, Network Analysis and Experimental Verication Used to Explore the Mechanism of Yangyin Yiqi Huoxue Recipe in Treating Sjogren’s Syndrome

Purpose: To explore the main active ingredients and mechanism of Yangyin Yiqi Huoxue (YYYQHX) Recipe in treating Sjogren’s syndrome. Methods: The main active ingredients of Yangyin Yiqi Huoxue(YYYQHX) Recipe were obtained through UPLC-LTQ-Orbitrap-MS n research technical analysis. Subsequently, the main active ingredients were analyzed by network pharmacology to explore their important targets and biological processes. Finally, the important target M3 muscarinic acetylcholine receptor (CHRM3) gene was veried by cytological experiments. Results: We identied 41 active ingredients in the YYYQHX Recipe, and identied 26 potential targets through network pharmacology methods. According to the network analysis and literature research, CHRM3 is one of the main targets of YYYQHX in the treatment of Sjogren’s syndrome. We observed the effect of YYYQHX on the CHRM3 and aquaporin 5(AQP5) genes using cytological experiments. The results showed that YYYQHX signicantly increased the expression of CHRM3 and AQP5 in submandibular gland cells. This may be the mechanism of YYYQHX Recipe in improving symptoms such as dry mouth in Sjogren’s syndrome. Conclusions: This study provides a strategy for understanding the mechanism of action of YYYQHX in treating Sjogren’s syndrome by combining UPLC-LTQ-Orbitrap-MS research, network pharmacology, and experimental validation. of AQP5 in the salivary gland epithelium of mice with experimental SS was decreased or absent, and the expression of AQP5 increased in the myoepithelial cells. These results indicate that the distribution and content of AQP5 are important factors that determine the transcellular transport of water. AQP5 the complex components and pharmacological mechanism of action of Yangyin Yiqi Huoxue Recipe, and identies potential therapeutic targets, which could provide a theoretical basis for the research and development of new drugs for the treatment of SS.


Background
Sjogren's syndrome (SS) is a chronic systemic autoimmune disease. The main organs involved are the salivary glands and lacrimal glands.
The symptoms are dry mouth and eyes, which seriously affects the quality of life of patients [1] . In the clinical practice of treating SS, it has been found that traditional Chinese medicine (TCM) can not only effectively improve patients' symptoms such as dry mouth and eyes and improve their quality of life, but it also has the advantages of a signi cant curative effect and few side effects [2,3] .
Yangyin Yiqi Huoxue Recipe (YYYQHX Recipe) is a TCM formula used in clinical practice, combining nine kinds of Chinese herbal medicine (Pseudostellariae Radix, Paeoniae Radix Alba, Polygonati Rhizoma, Fructus Ligustri Lucidi, Rhizoma Dioscoreae, Schisandrae Chinensis Fructus, Mume Fructus, Rhodiola rosea L., and Herba Sarcandrae). In clinical practice, we have found that treatment with YYYQHX is effective in treating SS. It not only improves the condition and relieves symptoms such as dry mouth, but also regulates the patient's Th1/Th2 immune balance and reproductive hormone-endocrine-immune function, and improves the quality of life [4,5] . Non-human animal experiments also showed that YYYQHX Recipe can reduce the amount of water drunk by NOD (Non-obese Diabetic) mice, increase saliva secretion, and reduce the pathological damage of submandibular gland tissue [6,7] .
In this study, UPLC-LTQ-Orbitrap-MS n research, network pharmacology and experimental veri cation were applied to investigate the therapeutic mechanism of YYYQHX Recipe in the treatment of SS. Speci cally, UPLC-LTQ-Orbitrap-MS research technology was used to analyze the main active ingredients in YYYQHX Recipe. Subsequently, the main active ingredients were analyzed by network pharmacology to explore their important targets and biological processes. In addition, the important targets were veri ed by cytological experiments to verify the pharmacological effects of YYYQHX Recipe on SS (Fig. 1). These results should clarify the therapeutic mechanism of TCM at the molecular level and improve the effectiveness and speci city of clinical treatment.
Mass spectrometry conditions: UPLC-Triple-TOF 5600 + Time-of-Flight LC/MS: negative ion scanning mode; scanning range: m/z 100-1500; atomizing gas (GS1): 55 psi; atomizing gas (GS2): 55 psi; curtain air (CUR): 35 psi; ion source temperature (TEM): 550 °C (negative); ion source voltage (IS): − 4500 V (negative); primary scan: declustering voltage (DP): 100 V; focusing voltage (CE): 10 V; secondary scan: TOF MS Product Ion IDA mode was used to collect mass spectrum data; the collision-induced dissociation(CID)nergy was − 20, − 40 and − 60 V; before sample injection, a calibrant delivery system(CDS) pump was used for mass axis calibration to ensure that the mass axis error was less than 2 ppm. The exact mass-to-charge ratio (m/z) was used to provide a reference for analysis and identi cation. During the identi cation process, the primary mass spectrum data comparison (ΔPPM < 10) was performed to identify possible compounds. On this basis, the compounds were further con rmed through secondary mass spectrometry analysis and literature search and comparison.

Targets of action of active compounds of Yangyin Yiqihuoxue Recipe and disease targets of SS
The active compounds obtained above were used to obtain prediction targets through the SwissTargetPrediction database (http://www.swisstargetprediction.ch/) and the SEA database (http://sea.bkslab.org/). In the Therapeutic Target Database (http://db.idrblab.org/ttd/), Drugbank Database (http://www.drugbank.ca), and Disgenet Database (http://www .disgenet.org), "Sjogren syndrome" was used as the search term to retrieve the disease target of SS; only one duplicate target was retained, and that target was taken to be the target related to SS.

Network construction and analysis
Mapping the target of the selected active compound with the disease target of SS and taking the intersection, the resulting target was the target of the YYYQHX Recipe activating a blood compound acting on the disease SS. The cytoscape software was used to construct the active compound of Yangyin Yiqi Huoxue Recipe and its target network for SS disease.

Gene ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) signal pathway analysis
Based on the targets obtained in the above process, the human genome annotation database DAVID 6.8 (http://david.ncifcrf.gov/) was used to conduct GO enrichment analysis and KEGG signal pathway analysis, screening with P < 0.05.

CCK8 detects cell proliferation
The HSG cell lines were randomly divided into a blank group, phosphate buffered saline (PBS) group, Yangyin Yiqi Huoxue Recipe (YYYQHX) group, and hydroxychloroquine (HCQ) group. After the above-mentioned drugs were used for 12, 24, and 48 h, a CCK-8 kit was used to calculate the cell survival rate.

Quantitative PCR(q-PCR) detection
The HSG cell lines were randomly divided into a blank group, PBS group, YYYQHX group, and hydroxychloroquine HCQ group. After 12, 24, and 48 h of drug action, q-PCR was performed to detect the levels of expression of the CHRM3 and AQP5 genes.

Data processing
Data were presented as mean ± SD and analyzed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). One-way analysis of variance (ANOVA) was used, and a P-value < 0.05 was considered statistically signi cant.

Part 1 3.1 Identi cation of active compounds of Yangyin Yiqi Huoxue Recipe based on the UPLC-LTQ-Orbitrap-MS platform
Based on the above sample processing methods and analysis conditions, the Yangyin Yiqi Huoxue Recipe was analyzed. In the positive and negative ion mode, 41 active compounds were screened from Yangyin Yiqi Huoxue Recipe, as shown in Fig. 2 and Table 1. From Therapeutic Target Database, Drugbank Database, Disgenet Database, using "Sjogren syndrome" as the search term to search for the relevant targets of SS, only one duplicate target was retained, and a total of 123 disease targets were obtained.
In the SwissTargetPrediction database and the SEA database, the above 41 active compounds were obtained as predicted targets; Finally, the disease targets of SS and the predicted targets of the active compounds of Yangyin Yiqi Huoxue Recipe were mapped to each other (only one repeated target remained), and 26 effect targets of Yangyin Yiqi Huoxue Recipe in the treatment of SS were obtained, as shown in Fig. 3 and Table 2. Table 2 Network Statistics of 26 effect targets

Network construction and analysis
The drug-active ingredient-target point network was constructed using Cytoscape software. As shown in Fig. 2, there were 63 nodes and 175 edges, of which yellow indicates Yangyin Yiqi Huoxue Recipe, orange indicates the 41 active compounds, and green indicates the 26 action targets. The edges represent the interaction of active ingredients with SS disease targets.
Each active ingredient could act on multiple targets, and each target was also related to multiple active compounds. This showed that Yangyin Yiqi Huoxue Recipe has a common effect on different compounds when exerting pharmacological effects.

GO enrichment analysis and KEGG signal pathway analysis
The human genome annotation database DAVID 6.8 was used to carry out GO enrichment analysis and KEGG signal pathway analysis on the 26 target points obtained above, using P < 0.05 as the screening condition to screen out biological processes and pathways with a large number of enriched genes. Compared with the NC group, when HCQ was used to treat the cells for 24 h, each dose group showed no cytotoxicity. When the drug action time was prolonged, the cell survival rate started to be lower than 90% at the concentration of 0.28 mg/ml at 48 h, and under the condition of HCQ action for 72 h, the cell viability in the ve HCQ dose groups was signi cantly reduced (P < 0.01): the higher the dose, the lower the cell survival rate.

q-PCR detection
The test results are shown in the Fig. 7. Compared with the control group, YYYQHX Recipe (10 mg/ml) up-regulated the levels of expression of CHRM3(P < 0.05)and AQP5 (P < 0.01). The HCQ group (3 mg/ml) also showed up-regulation of the expression of AQP5 (P < 0.01), but had no signi cant effect on CHRM3.

Discussion
SS is a chronic autoimmune disease. As with most autoimmune diseases, the etiology of Sjögren's syndrome is not yet fully understood.
There is currently no speci c medicine in the treatment of SS [8] . Hydroxychloroquine is a highly safe immunosuppressant with few side effects. It is the drug of choice when SS is mild to moderate. It can relieve clinical symptoms such as dry mouth, dry eyes, joint pain, arthritis, skin lesions and fatigue to a certain extent [9] . When the patient has severe organ manifestations, the treatment requires the use of large doses of methylprednisolone and cyclophosphamide, which have been proven to be effective [1] .
Traditional Chinese medicine has a long history and good clinical effects in treating diseases such as SS. Traditional Chinese medicine with the characteristics of holistic treatment has attracted increasing attention worldwide because of its satisfactory clinical effects [10,11] . As the main body and main form of Chinese medical practice, Chinese herbal medicine involves a variety of herbal ingredients and hundreds of chemical components [12] . Therefore, it is di cult to understand fully its effective ingredients and mechanisms of action, which depend on the overall interaction between all the ingredients.
In recent years, mass spectrometry technology has been widely used in the study of chemical components of traditional Chinese medicine, activity screening, and the study of small molecular compounds in metabolomics, and has played a huge role in elucidating the material basis and mechanisms of action of traditional Chinese medicine [13,14] .
In this study, we used UPLC-LTQ-Orbitrap-MS n technology to analyze a total of 41 active ingredients in Yangyin Yiqi Huoxue Recipe, combined with network pharmacological analysis. There appear to be 26 targets of Yangyin Yiqi Huoxue Recipe for treating SS. Among them, the target gene CHRM3 was most prominent. Through literature research, it was found that CHRM3 is closely related to the pathogenesis of SS. Therefore, CHRM3 was determined to be an important target for cell experiment veri cation.
Decreased saliva secretion is one of the important clinical manifestations of SS, and CHRM3 is closely related to saliva secretion [15,16] .
Studies have shown that M3 receptors are the most abundantly expressed subtype in the submandibular glands of different species such as humans, rats, mice, and rabbits, and are mainly distributed on acinar cells and vascular endothelial cells [15] .
The development of CHRM3 knockout mice is delayed after weaning, and intraperitoneal injection of the mAChR agonist pilocarpine caused little stimulation of salivation [17] . Studies have demonstrated that the saliva secretion of CHRM3 knockout mice is about 90% less than that of wild-type mice, and the knockout mice need to drink continuously to supplement the lack of saliva when eating, while M1 receptor gene knockout mice have no such phenotype [18,19] . This suggests that CHRM3 is the main gene involved in regulating saliva secretion.
AQP5 is an important protein that mediates the rapid transcellular transport of water in salivary gland cells [20] . AQP5 contributes to salivary secretion in patients with SS [21] . Abnormal distribution of AQP5 in labial salivary glands is associated with poor saliva secretion in patients with SS, including patients with the complication neuromyelitis optica. In the submandibular glands (SMG) of patients and mice with diabetes, the expression of AQP5 is reduced, which leads to the dysfunction of diabetic SMG, and saliva production decreases [21] .
There are also theories that the abnormal subcellular distribution and transport of AQP5 may be one of the important mechanisms of the pathogenesis of SS [22] . In the submandibular acinars of the non-obese diabetic(NOD) mice with in ammatory in ltration, the expression of AQP5 in the apical plasma membrane is decreased and the expression in the basement membrane is increased. Konttinen et al. [23] found that the expression of AQP5 in the salivary gland epithelium of mice with experimental SS was decreased or absent, and the expression of AQP5 increased in the myoepithelial cells. These results indicate that the distribution and content of AQP5 are important factors that determine the transcellular transport of water.
We observed the levels of expression of the CHRM3 and AQP5 genes in submandibular gland cells under the action of Yangyin Yiqi Huoxue Recipe and hydroxychloroquine in experimental cell studies. From the experimental results, we found that the levels of expression of the CHRM3 and AQP5 genes in submandibular gland cells were signi cantly increased under the action of Yangyin Yiqi Huoxue Recipe. The hydroxychloroquine group showed up-regulation of the expression of the AQP5 gene, but no obvious up-regulatory effect on CHRM3. This experimental result con rmed that CHRM3, obtained from the previous network pharmacological analysis, is one of the important targets of the effect of Yangyin Yiqi Huoxue Recipe. The CHRM3 and AQP5 genes are closely involved in the secretion of saliva. This may be the mechanism by which Yangyin Yiqi Huoxue Recipe can improve symptoms such as dry mouth in SS.

Conclusions
In this study, using a combination of UPLC-LTQ-Orbitrap-MS n technology, network pharmacological analysis and experimental veri cation, a comprehensive strategy was adopted to illustrate the active ingredients and molecular mechanisms of Yangyin Yiqi Huoxue Recipe in the treatment of SS. Using UPLC-LTQ-Orbitrap-MS n technology, 41 kinds of active ingredient were determined in the herbal database and used for network pharmacological analysis to allow interpretation of the active ingredients in the whole prescription more objectively. Based on the results of network pharmacological analysis and literature research, this study identi ed CHRM3 as the research target. Through experimental veri cation of the target CHRM3, the mechanism of Yangyin Yiqi Huoxue Recipe in treating SS was elucidated from the perspective of "drugcomponent-target". However, because of the small number of targets, research on signal pathways could not be presented here. This study reveals the complex components and pharmacological mechanism of action of Yangyin Yiqi Huoxue Recipe, and identi es potential therapeutic targets, which could provide a theoretical basis for the research and development of new drugs for the treatment of SS.   KEGG signal pathway analysis on the 26 target points Figure 6 CCK-8 detects cell proliferation in YYYQHX Recipe groups and HCQ Groups