Investigation of the Mechanism of Shengmai Injection on Sepsis by Network Pharmacology Approaches

Shengmai injection (SMI) contains Ginsen Radix et Rhizoma Rubra, Ophiopogon japonicus, and Schisandrae Chinensis Fructus. It is used as a supportive herbal medicine in the management of sepsis, systemic inflammatory response syndrome, and septic or hemorrhagic shock. An UPLC method was established to identify and evaluate SMI fingerprints. Fingerprint similarities of 9 batches of SMI were compared. The network platform, “TCM-components-core targets-key pathways,” was established, and the mechanism of SMI in the treatment of sepsis was investigated. The similarity of 9 batches of SMI fingerprints was greater than 0.91. 44 peaks were selected as the common peaks, of which 11 peaks were identified. KEGG functional pathway analysis showed SMI was mainly involved in the pathways of cancer, cell cycle, and p53 signaling, suggesting SMI protects multiple organs via regulating immunity, inflammation, apoptosis, and energy metabolism. GO enrichment analysis showed active SMI components regulated various biological processes and altered the pathophysiology of sepsis. The interplays between SMI and multiple energy metabolism signaling cascades confer protection from life-threatening multiple organ failure in sepsis.


Introduction
Sepsis is a deregulated body response to infection, triggering inflammatory reactions that can cause systemic symptoms and damage multiple organs. Release of cytokines mediates uncontrolled inflammatory cascades that result in dysfunction and failure of multiple major organs and septic shock [1,2]. Managing infection is the most critical strategy for sepsis therapy. However, these treatments could cause various side effects [3]. Clinically, sepsis is managed with early use of antibiotics and glucocorticoids. Traditional Chinese medicine, including Xuebijing injection [2,4], Shenfu injection [5], and SMI [6], provides supportive effects in sepsis treatment.
Shengmai injection (SMI) origins from the ancient prescription of Chinese medicine Shengmaiyin; it contains Ginsen Radix et Rhizoma Rubra, Ophiopogon japonicus, and Schisandrae Chinensis Fructus. It invigorates Qi, nourishes Yin, and promotes blood circulation. SMI is used as add-on for supportive treatment in managing patients with sepsis, systemic inflammatory syndrome, and septic or hemorrhagic shock [7][8][9]. e frequency of adverse events associated with SMI is low [10,11]. ere have been few reports on the evaluation of SMI effective components and their underlying mechanisms despite they are used as supportive interventions for sepsis treatment. It is a compound with multiple components targeting multiple molecular networks; exploring its complex antisepsis mechanism in a suitable model is of great importance for sepsis treatment [12]. Network pharmacology is a useful tool for systemic investigation of the mechanisms of multiple component drugs [13,14]. Its approach has been used to study "compound-protein/gene-disease" pathways which reveal complexities among drugs, biological systems, and diseases from a network perspective. Network pharmacology provides insights into the complex interrelationships between the active ingredients of traditional Chinese compounds and molecular mechanisms [15].
We established a fingerprint method to detect and represent chemical information of SMI. We mapped potential targets of SMI bioactive ingredients that may regulate the progress of sepsis using a network pharmacology approach. Our findings shed light on further understanding of the mechanisms of SMI in treating complex diseases such as sepsis.

UPLC Conditions.
e analyte was separated by a Waters Acquity UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column. e mobile phases used were solvent A (acetonitrile) and solvent B (water) with gradient elution (Table 1). e analysis was carried out at a flow rate of 0.3 mL/min. e column temperature was set to 40°C. UV detection wavelength was over the range of 190 to 400 nm. 5 μL of the sample was injected. 210 nm was selected as the extraction wavelength of the fingerprints.

Precision of the Method.
Method precision was determined by analyzing the same sample SMI (S1, 16120101005) five consecutive times in a day. e peak of schisantherin A was used as the reference peak. Relative standard deviation (RSD) was calculated from the relative peak area (RPA) or relative retention time (RRT) of each characteristic peak.

Sample Stability.
Sample stability was evaluated using the same SMI (S1, 16120401005) after 0, 2, 4, 6, 8, 12, and 24 hours. e peak of schisantherin A was used as the reference peak. RSD was calculated from RPA or RRT of each peak to the reference peak from the chromatographic profiles of samples.
2.6. Repeatability. Repeatability was evaluated by analyzing six independently prepared SMI samples. e peak of schisantherin A was used as the reference peak. RSD was calculated from RPA or RRT of each peak to the reference peak from the chromatographic profiles of samples.

Network Construction.
Data acquisition and processing were done in databases which include SciFinder and TCMSP. Additionally, PubChem CID for each active ingredient of SMI was obtained from PubChem. We used SMILES format in STITCH chemical association networks and obtained the interaction complex between SMI bioactive components and the potential target protein in humans. Using HPO as a tool, we annotated and analyzed the core protein targets that participate in sepsis. e primary as well as predicted interactions between SMI target proteins and proteins involved in sepsis were analyzed in the STRING  Evidence-Based Complementary and Alternative Medicine database. We collected core proteins that are highly associated with sepsis, while proteins with low correlation were filtered out [16]. e molecular interplays between SMI key targets and sepsis proteins were visualized in the Cytoscape platform. We calculated the degree, betweenness, and closeness of the targets; proteins with topological parameters greater than the corresponding median values were considered as major hits. e selected proteins were validated in  the OMIM database to establish protein-disease association and construct "SMI Targets-Sepsis Targets" network.

Prediction of the SMI-Antisepsis Mechanism.
A list of the selected top 20 key proteins was uploaded to the DAVID database for functional annotation and enrichment analysis to obtain the main pathways and network distribution that confer potential mechanisms for SMI treatment. Only pathways with p < 0.05 were considered for mechanism prediction.

Establishment of SMI Fingerprints and the Results of Methodological Evaluation.
e RSDs of RPA and RRT for precision, repeatability, and sample stability were lower than 3%, respectively. e results showed that the fingerprint method developed for analysis of SMI is reliable and applicable. Figures 1 and 2 show the UPLC chromatogram fingerprints of 9 batches of SMI.

Similarity of Fingerprints of 9 Batches of SMI.
e similarities of all chromatographic patterns among the samples ( Table 2) were calculated using software "Chromatographic Fingerprints of Traditional Chinese Medicine, version: 2004A." e similarities of 9 SMI batches were greater than 0.91. erefore, our method was precise, stable, reproducible, and reliable.

Acquisition of Known
erapeutic Sepsis Targets. Sepsis targets were collected from the HPO database. e keyword "sepsis" was used to search known therapeutic targets for sepsis in humans [21]. A total of 58 sepsis targets (Table 4) were acquired from the HPO database, and targets were further verified in the NCBI database.

Results of Network Construction.
e putative targets of SMI active ingredients and sepsis disease targets were determined based on the protein-protein interactions [22]. e interplays amongst SMI targets, known therapeutic targets for sepsis, and interactional human targets were combined to construct the network. e network illustrates the relationship between SMI targets and sepsis targets. e overall interaction network ( Figure 5) was visualized using Cytoscape (sepsis targets in red circles and SMI targets in blue squares); the larger a node, the more targets it contains and more important in sepsis management. Targets with higher values of "degree," "betweenness," "closeness," and "coreness" (above the median value of all the network nodes) were identified [23,24]. Targets which might play unimportant roles in the network according to the topological features were discarded [25]. Median value of "degree," "betweenness," and "closeness" was 19, 0.014, and 0.449, respectively. Top twenty proteins were selected as key sepsis therapeutic targets ( Figure 6 and Table 5), including ABL1, CCND1, CDK family (CDK1, CDK2, CDK6, and CDKN1B), RB1, HSP90AA1, SMARCA4, RBL2, CTNNB1, MDM2, SP1, LRRK1, BTK, PIK3R1, TMPRSS11D, ACTG2, CD79A, and RET.
Sepsis causes life-threatening organ dysfunction due to a host's complex systemic inflammatory response to infection [26]. In the present study, we identified core proteins that may play important roles in SMI-supportive treatment in sepsis. ABL1 is a tyrosine-protein kinase which is important for cell growth and survival, cytoskeleton remodeling in response to extracellular stimuli, autophagy, and apoptosis [27][28][29]. It also regulates multiple pathological signaling cascades during infection that alter vascular permeability and the endothelium barrier in inflammation [30].
Cyclin-dependent kinase 1 (CDK1) is a member of the Ser/ r protein kinase family. Its kinase activity is controlled by cyclin [31,32]. CCND1/CDK4 and CDK2 are critical for G1/S phase transition. It has been shown rat kidney injury is associated with G1 phase arrest in cecal ligation and puncture-(CLP-) induced sepsis, while upregulation of CCND1/CDK4 and CCNE/CDK2 activates Rb leading to revival of cell cycle progress and recovery of kidney function  30  ACTG2  31  CYP4F22  32  AK2  33  CD79A  34  CD79B  35  NCF2  36  IKZF1  37  ALOXE3  38  NCF4  39  SERAC1  40  LIPN  41  CHD7  42  IGLL1  43  RET  44  PLEC  45  CTNNB1  46  ECE1  47  ADA  48  IL2RG  49  ITGB4  50  GDNF  51  MUT  52  ALOX12B  53  EDN3  54  EDNRB  55  IL7R  56  FERMT3  57  TFRC  58  CYBA   6 Evidence-Based Complementary and Alternative Medicine  Evidence-Based Complementary and Alternative Medicine 7 cell cycle arrest occurs in sepsis, and drugs that regulate cell cycle proteins may be a means to rescue organ injury [35]. In addition, the targets of SMI are more involved in DNA replication and transcription; for example, MDM2, E3 ubiquitin ligase, mediates ubiquitination and degradation of p53. It mediates apoptosis in organ injury and malignant transformation [36]. SMI may inhibit MDM2 and keep p53 active; therefore, it promotes cells staying in the G1/G2 phase and alleviates cell injury in sepsis.
Molecular chaperone heat-shock protein (HSP 90) is extensively expressed by cells, and its expression increases upon stimulation [37]. HSPs are associated with multiple organ failure in sepsis [38]. In the vast immune response in sepsis, stressed cells release HSPs that are regarded as "danger signal" to neighboring and immune cells [39]. HSP90-α has been shown to interact with about 200 client proteins, including signal proteins in the inflammatory pathway such as NF-κB, Akt, and IKK, to interfere inflammation [40][41][42]. Moreover, HSP90-α, as abundant "chaperone," is one of the main mediators that activates bacteria lipopolysaccharide. It interacts with proteins in the PI3K/Akt pathway and is essential in promoting the immune response and improving host defense to pathogens. Inhibition of HSP90-α prevents severe sepsis-associated acute lung injury; therefore, block HSP90-α offers a novel treatment for lung injury in sepsis [43][44][45].
BTK (tyrosine-protein kinase) is a component of the tolllike receptor (TLR) pathway and plays important roles in innate and adaptive immunity. Key target CD79A is required for efficient differentiation of pro-and pre-B-cells. It cooperates with CD79B and bounds to the B-cell antigen receptor complex (BCR) for initiation of the signal transduction cascade. It is pivotal in regulating immunity and inflammation [46]. Network pharmacology revealed SMI represses BTK expression/activation, blocks signals through multiple pathways (TLR, B-cell antigen receptor signal, and apoptosis), and consequently ameliorates cell apoptosis and organ injury. SMI acts as a whole, and each formula has its corresponding targets/syndromes; thus, SMI prescription acts on multiple key targets, and the network pharmacology study of SMI provides insights into understanding its fundamental mechanisms.

KEGG and GO Analysis.
To cluster the biological functions of SMI and its targets, data were uploaded to KEGG, and results revealed SMI active formulae target pathways including cancer, cell cycle, p53, B-cell receptor, and ErbB pathways ( Table 6). SMI regulates the interplay and synergy among the pathways of immunity, inflammation, and apoptosis to protect cellular and organ injury in sepsis. p53 pathway regulates mitochondrial fission and mitochondrial biogenesis via AMPK, and it alters PKM2dependent glycolysis. Global deletion of PKM2 results in systemic inflammation in mice [47]. Our GO analysis (Table 7) showed that SMI ingredients regulate multiple  Evidence-Based Complementary and Alternative Medicine biological processes including cell cycle, energy metabolism, cellular signal transduction, transcription regulation, and immunity development. Our data indicate the putative role of SMI in alleviating systemic inflammation and deregulating immunity in the host; moreover, it regulates energy utilization and promotes energy homeostasis and therefore ameliorates multiple organ failure associated with sepsis. It is also in agreement with the idea of SMI used in the early phase of sepsis.

Conclusion
An UPLC method was developed for analysis of SMI fingerprints. Forty-four peaks were selected as common peaks, of which 11 peaks were identified. e consistency in the chromatograms of 9-batch samples reflects the presence of similar chemical constituents (similarities greater than 0.91). e technique was proven to be useful in SMI quality control. A total of 9 active components of SMI target 20 key proteins including ABL1, CDK, HSP90, BTK, PIK3R1, and CD79A. ese proteins are enriched in cell cycle, p53 signaling pathway, B-cell receptor signaling pathway, and ErbB pathway. It is likely that the pharmacological mechanisms of SMI in sepsis treatment are of multiple dimensions that are associated with regulation of cell cycle, energy metabolism, cellular signal transduction, transcription regulation, and immunity development. Further experiments are needed to validate our prediction.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.